Anti-HLA-G antibodies and use thereof

ABSTRACT

The present invention relates antibodies that bind to human HLA-G, multispecific antibodies thereof, their preparation, formulations and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of European Application No.21203272.6, filed Oct. 18, 2021, and European Application No.20214951.4, filed Dec. 17, 2020, which are incorporated herein byreference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 15, 2021, is namedP36618-US_Sequence Listing_ST25.txt and is 145,691 bytes in size.

FIELD OF INVENTION

The present invention relates to anti-HLA-G antibodies, theirpreparation, formulations and methods of using the same.

BACKGROUND OF THE INVENTION

The human major histocompatability complex, class I, 6, also known ashuman leukocyte antigen G (HLA-G), is a protein that in humans isencoded by the HLA-G gene. HLA-G belongs to the HLA nonclassical class Iheavy chain paralogues. This class I molecule is a heterodimerconsisting of a heavy chain and a light chain (beta-2 microglobulin).The heavy chain is anchored in the membrane but can also beshedded/secreted.

-   -   The heavy chain consists of three domains: alpha 1, alpha 2 and        alpha 3. The alpha 1 and alpha 2 domains form a peptide binding        groove flanked by two alpha helices. Small peptides        (approximately 9-mers) can bind to this groove akin to other MHC        I proteins.    -   The second chain is beta 2 microglobulin which binds to the        heavy chain similar to other MHC I proteins.

For HLA-G there exist 7 isoforms, 3 secreted and 4 membrane bound forms(as schematically shown in FIG. 1).

HLA-G can form functionally active complex oligomeric structures(Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729). Disulfide-linkeddimers are formed between Cys 42 of two HLA-G molecules. (ShiroishiMetal., J Biol Chem 281 (2006) 10439-10447. Trimers and Tetramericcomplexes have also been described e.g. in Kuroki, K et al. Eur JImmunol. 37 (2007) 1727-1729, Allan D. S., et al. J Immunol Methods. 268(2002) 43-50 and T Gonen-Gross et al., J Immunol 171 (2003)1343-1351).

HLA-G is predominantly expressed on cytotrophoblasts in the placenta.Several tumors (including pancreatic, breast, skin, colorectal, gastric& ovarian) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791;Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). The expressionhas also been reported to be associated with pathological conditionslike inflammatory diseases, GvHD and cancer. Expression of HLA-G hasbeen reported to be associated with poor prognosis in cancer. Tumorcells escape host immune surveillance by inducing immunetolerance/suppression via HLA-G expression.

Overview polymorphisms HLA family HLA-A: 2579 seqs HLA-A: 3283 seqs{close oversize brace} classical class I MHC HLA-C: 2133 seqs HLA-E: 15seqs HLA-F: 22 seqs {close oversize brace} non-classical class I MHCHLA-G: 50 seqs

HLA-G shares high homology (>98%) with other MHC I molecules, thereforetruly HLA-G specific antibodies with no crossreactivity to other MHC Imolecules are difficult to generate.

Certain antibodies which interact in different ways with HLA-G weredescribed previously: Tissue Antigens, 55 (2000) 510-518 relates tomonoclonal antibodies e.g. 87G, and MEM-G/9; Neoplasma 50 (2003) 331-338relates to certain monoclonal antibodies recognizing both, intact HLA-Goligomeric complex (e.g. 87G and MEM-G9) as well as HLA-G free heavychain (e.g. 4H84, MEM-G/1 and MEM-G/2); Hum Immunol. 64 (2003) 315-326relates to several antibodies tested on HLA-G expressing JEG3 tumorcells (e.g. MEM-G/09 and -G/13 which react exclusively with nativeHLA-G1 molecules. MEM-G/01 recognizes (similar to the 4H84 mAb) thedenatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizesselectively denatured HLA-G1, -G2, and -G5 isoforms; Wiendl et al Brain2003 176-85 relates to different monoclonal HLA-G antibodies as e.g.87G, 4H84, MEM-G/9.

The above publications report antibodies, which bind to human HLA-G orthe human HLA-G-β2M MHC complex. However, due to the high polymorphismand high homology of the HLA family most of the antibodies lack eithertruly specific HLA-G binding properties and often also bind orcrossreact with other HLA family members (either as MHC complex with β2Mor in its β2M-free form) or they simply do not inhibit binding ofHLA-Gβ2M MHC complex to its receptors ILT2 and/or ILT4 (and are regardedas non-antagonistic antibodies).

WO2019/202040 relates to HLA-G antibodies including antibody HLA-G-0090.WO 2019/202041 relates to multispecific HLA-G antibodies includingantibody HLA-G-0090.

SUMMARY OF THE INVENTION

The invention described herein provides an antibody that binds to humanHLA-G comprising

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:24.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:26.

Such antibodies have highly valuable properties like their bindingproperties, their high specificity towards HLA-G with no crossreactivityto HLA-A and HLA MHC I complexes from other species. They can bind toHLA-G on cells and inhibit ILT2 and/or ILT4 binding to HLA-G expressedon these cells. They have been generated from the HLA-G antibodyHLA-G-0090.

As the HLA-G antibody HLA-G-0090 described in WO 2019/202040 comprises aglycosylation site in one of the CDRs (CDR-L1 which comprises a NSSmotif at amino acids 31, 32 and 33 of the light chain (LC)), its bindingproperties are impacted by the N-glycosylation which constitutes apotential developability liability.

A homology model of the variable region of HLA-G-0090 indicated thatlight chain (LC) positions 31 to 33 are highly solvent accessible.Furthermore, the side chains of N31 and S32 are predicted to pointinwards, in the direction of CDR-H3, making them likely candidates forbeing part of the antibody paratope. In fact, a number of publishedantibody-antigen X-ray complex structures document these residues to beundergoing chemical interactions with the antigen. Therefore, the riskof worsening the binding affinity of the antibody by introducingmutations at LC positions 31-33 was high. Therefore various variants ofantibody HLA-G-0090 with mutations on LC positions 31, 32, and 33 weredesigned from which however most variants worsened binding properties orexpressability. Surprisingly it was found that among these variousvariants of HLAG-0090 in which the glycosylation site was removed onlythe two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A show evenimproved binding properties, good expressability and stability, whileshowing no more N-glycosylation at the CDR-L1 of the LC (so no Fabglycosylation could be detected). As all recently approvedpharmaceutical antibody products are produced in mammalian cells,especially CHO cells (see e.g. Walsh G., Nature Biotech (2018)1136-1145), providing an antibody without glycosylation sites in thebinding region (VH and VL and especially the CDRs) represents a valuableadvantage, as these antibodies can readily be used for production inmammalian expression systems without the risk of (at least partially)impairing the binding properties by glycosylation.

In a further embodiment the HLA-G antibody of the present inventioncomprises a Fc domain of human origin. In one embodiment the Fc domainis of the IgG isotype, in one preferred embodiment of the IgG1 isotype.

In a further embodiment the HLA-G antibody of the present invention is abispecific antibody, in particular an a bispecific antibody that bindsto human HLA-G and to human CD3, comprising a first antigen bindingmoiety that binds to human HLA-G and a second antigen binding moietythat binds to human CD3.

In one embodiment the HLA-G antibody of the present invention is abispecific antibody that binds to human HLA-G and to human CD3,comprising a first antigen binding moiety that binds to human HLA-G anda second antigen binding moiety that binds to human CD3,

wherein the first antigen binding moiety that binds to human HLA-Gcomprises

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6;

and wherein the second antigen binding moiety that binds to a T cellactivating antigen binds to human CD3 comprises

C) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:52, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:53, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:54; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:55; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:56 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:57, or

D) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:60, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:61, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:62; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:63; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:64 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:65, or

E) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:68, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:69, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:70; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:71; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:72 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:73.

One embodiment of the invention is such bispecific antibody,

wherein the first antigen binding moiety

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26,and wherein the second antigen binding moiety

C) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:58 and a VL domain comprising the amino acid sequence of SEQ IDNO:59; or

D) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:66 and a VL domain comprising the amino acid sequence of SEQ IDNO:67; or

E) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:74 and a VL domain comprising the amino acid sequence of SEQ IDNO:75.

One embodiment of the invention is such bispecific antibody,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:58and a VL domain comprising the amino acid sequence of SEQ ID NO:59.

One embodiment of the invention is such bispecific antibody,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:66and

-   -   a VL domain comprising the amino acid sequence of SEQ ID NO:67.

One embodiment of the invention is such bispecific antibody,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:74and a VL domain comprising the amino acid sequence of SEQ ID NO:75.

Such bispecific antibodies binding to human HLA-G and human CD3 show inaddition to the properties of the HLA-G-antibodies further valuableproperties like the induction of antibody mediated IFN gamma secretionby T cells on HLA-G expressing cells, T cell activation in the presenceof HLA-G expressing tumor cells, induction of T cell mediated tumor cellkilling on HLA-G expressing cells and in vivo anti-tumor efficacy andeven tumor regression in different cancer xenograft mouse models.

The invention provides an isolated nucleic acid encoding the antibody orbispecific antibody as described herein.

The invention provides a host cell comprising such nucleic acid.

The invention provides a method of producing an antibody comprisingculturing the host cell so that the antibody is produced.

The invention provides such method of producing an antibody, furthercomprising recovering the antibody from the host cell.

The invention provides an antibody produced by such an host cell wherethe host cell is eukaryotic.

The invention provides a pharmaceutical formulation comprising theantibody described herein and a pharmaceutically acceptable carrier.

The invention provides the antibody described herein for use as amedicament.

The invention provides the antibody described herein for use in treatingcancer.

The invention provides the use of the antibody described herein in themanufacture of a medicament. In one embodiment the medicament is fortreatment of cancer.

The invention provides a method of treating an individual having cancercomprising administering to the individual an effective amount of theantibody described herein.

DESCRIPTION OF THE FIGURES

FIG. 1: Different isoforms of HLA-G

FIG. 2: FIG. 2A: Schematic representation of the HLA-G molecule inassociation with β2M:

-   -   Schematic representation of the HLA-G wt molecule    -   FIG. 2B: Schematic representation of the HLA-G molecule in        association with β2M:    -   The KIR2DL4 and ILT2/4 interactions are extracted from crystal        structures: the HLA-G:ILT4 complex structure (PDB code: 2DYP).        The KIR2DL1 structure is taken from PDB code 1IM9        (KIR2DL1:HLA-Cw4 complex structure) and was positioned on HLA-G        by superposition of the HLA-Cw4 and HLA-G structures.    -   FIG. 2C: Schematic representation of the HLA-G molecule in        association with β2M:    -   Schematic representation of the HLA-G chimeric molecule that was        used as a counter antigen for the identification of specific        HLA-G binders. White dots represent surface residues that were        identified as unique for HLA-G. These residues were replaced by        a HLA consensus sequence in the chimeric molecule.    -   FIG. 2D: Structure of HLA-G molecule in association with certain        receptors: HLA-G structure in complex with given receptors such        as ILT4 and KIR2DL1. ILT4 structure (PDB code: 2DYP). The        KIR2DL1 structure is taken from PDB code 1IM9 (KIR2DL1: HLA-Cw4        complex structure) and was positioned on HLA-G by superposition        of the HLA-Cw4 and HLA-G structures. Receptors are shown in a        ribbon representation, HLA-G is shown in a molecular surface        representation. HLA-G residues that are unique or conserved in        other HLA paralogs are colored in white and gray, respectively.        Unique surface residues were replaced by a HLA consensus        sequence in the chimeric counter antigen.

FIG. 3: Schematic antibody-antigen binding assay principle—relativeactive concentration (RAC) of HLA-G antibodies for binding to HLA-G

FIG. 4A: Mass spectrum of N-glycosylation of HLA-G-0090 indicatesFab-glycosylation

FIG. 4B: Mass spectra of N-glycosylation of HLA-G-0090-VL-S32P andHLA-G-0090-VL-S33A: No Fab-glycosylation detectable

FIG. 5: exemplary FACS staining for anti-HLA-G antibodies HLA-G-0090,HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A (2 μg/ml) on SKOV3 cells (noHLA-G expression, JEG3 cells (expressing HLA-G) and SKOV3-HLA-G cells(SKOV3 cells transfected with HLA-G)

FIG. 6: Ability of the HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32Pand HLA-G-0090-VL-S33A to modify/inhibit the interaction and binding ofrecombinant soluble ILT2 (ILT2Fc domain fusion) to HLA-G naturallyexpressed on JEG3 tumor cells. JEG3 cells are preincubated/pretreatedwith HLA-G antibodies so that ILT2 binding to JEG3 cells isinhibited/blocked. Controls were carried out with JEG3 cells withoutHLA-G antibody pretreatment (only ILT2-Fc) and isotype antibody.

FIG. 7: Binding of bispecific anti-HLA-G/anti-CD3 T cell bispecific(TCB) antibody to CD3 expressed on T-cells by antibodies P1AF7977,P1AF7978 and P1AF7979

FIG. 8: Bispecific anti-HLA-G/anti-CD3 T cell bispecific (TCB)antibodies P1AF7977, P1AF7978 and P1AF7979 showed binding to JEG3 cellsand SKOV3 cells, transfected with HLA-G.

FIG. 9: Bispecific anti-HLA-G/anti-CD3 T cell bispecific (TCB) antibodymediated/induced IFN gamma secretion by T cells, by antibodies P1AF7977,P1AF7978 and P1AF7979

FIG. 10: Bispecific anti-HLA-G/anti-CD3 T cell bispecific (TCB)antibodies P1AF7977, P1AF7978 and P1AF7979 induced T cell mediatedcytotoxicity/tumor cell killing.

FIG. 11: In vivo anti-tumor efficacy of anti-HLA-G/anti-CD3 T cellbispecific (TCB) antibody P1AF7977 in humanized NSG mice bearing SKOV3human ovarian carcinoma transfected with recombinant HLA-G (SKOV3HLA-G), leading to tumor regression

FIG. 12: Dose-response study with anti-HLA-G/anti-CD3 T cell bispecific(TCB) antibody P1AF7977 in humanized NSG mice bearing human breastcancer PDX tumors (BC004). Strong tumor growth inhibition until tumorregression is observed in mice treated with different doses.

FIG. 13: Exemplary configurations of the bispecific antigen bindingmolecules of the invention. (A, D) Illustration of the “1+1 CrossMab”molecule. (B, E) Illustration of the “2+1 IgG Crossfab” molecule withalternative order of Crossfab and Fab components (“inverted”). (C, F)Illustration of the “2+1 IgG Crossfab” molecule. (G, K) Illustration ofthe “1+1 IgG Crossfab” molecule with alternative order of Crossfab andFab components (“inverted”). (H, L) Illustration of the “1+1 IgGCrossfab” molecule. (I, M) Illustration of the “2+1 IgG Crossfab”molecule with two CrossFabs. (J, N). Black dot: optional modification inthe Fc domain promoting heterodimerization. ++, −−: amino acids ofopposite charges optionally introduced in the CH1 and CL domains.Crossfab molecules are depicted as comprising an exchange of VH and VLregions, but may—in embodiments wherein no charge modifications areintroduced in CH1 and CL domains—alternatively comprise an exchange ofthe CH1 and CL domains.

FIG. 14: Induction of T cell activation by bispecificanti-HLA-G/anti-CD3 antibody P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035 inthe presence of SKOV3 HLAG cells.

FIG. 15: Relative binding activity of original and optimized CD3binders, CD3_(orig) and CD3_(opt) (=P035-093 (P035)), to recombinant CD3as measured by SPR in unstressed condition, after 14 d at 40° C. pH 6,or after 14 d at 37° C. pH 7.4 (IgG format).

FIG. 16: Binding of original and optimized CD3 binders, CD3_(orig) andCD3_(opt) (=P035-093 (P035)), to Jurkat NFAT cells as measured by flowcytometry (IgG format). Antibodies bound to Jurkat NFAT cells weredetected with a fluorescently labeled anti-human Fc specific secondaryantibody.

FIG. 17: Schematic illustration of the CD3 activation assay used inExample 8.

FIG. 18: Jurkat NFAT activation with original and optimized CD3 binders,CD3_(orig) and CD3_(opt) (=P035-093 (P035)) (IgG format). Jurkat NFATreporter cells were co-incubated with anti-PGLALA expressing CHO(CHO-PGLALA) cells in the presence of CD3_(orig) or CD3_(opt) (=P035-093(P035)) IgG PGLALA, or CD3_(opt) IgG wt as negative control. CD3activation was quantified by measuring luminescence after 24 h.

DETAILED DESCRIPTION OF THE INVENTION

When used herein, the term “HLA-G”, “human HLA-G”, “HLAG”, refers to theHLA-G human major histocompatibility complex, class I, G, also known ashuman leukocyte antigen G (HLA-G) (exemplary SEQ ID NO: 35). Typically,HLA-G forms a MHC class I complex together with β2 microglobulin (B2M orβ2m). In one embodiment HLA-G refers to the MHC class I complex of HLA-Gand β2 microglobulin. In one preferred embodiment HLA-G refers to thecell surface bound MHC class I complex of HLA-G and β2 microglobulin,also known as HLA-G1 (see FIG. 1 of this description and e.g. Blaschitzet al., Molecular Human Reproduction, 11(2005) 699-710, inter alia FIG.1)

As used herein, an antibody (either mono-, multi- or bispecific) orantigen binding moiety “binding to human HLA-G”, “specifically bindingto human HLA-G”, “that binds to human HLA-G” or “anti-HLA-G” refers toan antibody/antigen binding moiety specifically binding to the humanHLA-G antigen or its extracellular domain (ECD) with a binding affinityof a K_(D)-value of 5.0×10⁻⁸ mol/l or lower, in one embodiment of aKD-value of 1.0×10⁻⁹ mol/l or lower, in one embodiment of a KD-value of5.0×10⁻⁸ mol/l to 1.0×10⁻¹³ mol/l. In one embodiment the antibody bindsto HLA-G β2M MHC I complex comprising SEQ ID NO: 39)

The binding affinity is determined with a standard binding assay, suchas surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala,Sweden) e.g. using constructs comprising HLA-G extracellular domain(e.g. in its natural occurring 3 dimensional structure). In oneembodiment binding affinity is determined with a standard binding assayusing exemplary soluble HLA-G comprising MHC class I complex comprisingSEQ ID NO: 39.

HLA-G has the regular MHC I fold and consists of two chains: Chain 1consists of three domains: alpha 1, alpha 2 and alpha 3. The alpha 1 andalpha 2 domains form a peptide binding groove flanked by two alphahelices. Small peptides (approximately 9 mers) can bind to this grooveakin to other MHCI proteins. Chain 2 is beta 2 microglobulin (β2M) whichis shared with various other MHCI proteins.

HLA-G can form functionally active complex oligomeric structures(Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729). Disulfide-linkeddimers are formed between Cys 42 of two HLA-G molecules. (ShiroishiMetal., J Biol Chem 281 (2006) 10439-10447. Trimers and Tetramericcomplexes have also been described e.g. in Kuroki, K et al. Eur JImmunol. 37 (2007) 1727-1729, Allan D. S., et al. J Immunol Methods. 268(2002) 43-50 and T Gonen-Gross et al., J Immunol 171 (2003)1343-1351).HLA-G has several free cysteine residues, unlike most of the other MHCclass I molecules. Boyson et al., Proc Nat Acad Sci USA, 99: 16180(2002) reported that the recombinant soluble form of HLA-G5 could form adisulfide-linked dimer with the intermolecular Cys42-Cys42 disulfidebond. In addition, the membrane-bound form of HLA-G1 can also form adisulfide-linked dimer on the cell surface of the JEG3 cell line, whichendogenously expresses HLA-G. Disulfide-linked dimer forms of HLA-G1 andHLA-G5 have been found on the cell surface of trophoblast cells as well(Apps, R., Tissue Antigens, 68:359 (2006)).

HLA-G is predominantly expressed on cytotrophoblasts in the placenta.Several tumors (including pancreatic, breast, skin, colorectal, gastric& ovarian) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791;Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). The expressionhas also been reported to be associated with pathological conditionslike inflammatory diseases, GvHD and cancer. Expression of HLA-G hasbeen reported to be associated with poor prognosis in cancer. Tumorcells escape host immune surveillance by inducing immunetolerance/suppression via HLA-G expression.

For HLA-G there exist 7 isoforms, 3 secreted and 4 membrane bound forms(as schematically shown in FIG. 1). The most important functionalisoforms of HLA-G include b2-microglobulin (β2M)-associated HLA-G1 andHLA-G5. However, the tolerogenic immunological effect of these isoformsis different and is dependent on the form (monomer, dimer) of ligandsand the affinity of the ligand-receptor interaction.

HLA-G protein can be produced using standard molecular biologytechniques. The nucleic acid sequence for HLA-G isoforms is known in theart. See for example GENBANK Accession No. AY359818.

The HLA-G isomeric forms promote signal transduction through ILTs, inparticular ILT2, ILT4, or a combination thereof.

ILTs: ILTs represent Ig types of activating and inhibitory receptorsthat are involved in regulation of immune cell activation and controlthe function of immune cells (Borges, L., et al., Curr Top MicrobialImmunol, 244:123-136 (1999)). ILTs are categorized into three groups:(i) inhibitory, those containing a cytoplasmic immunoreceptortyrosine-based inhibitory motif (ITIM) and transducing an inhibitorysignal (ILT2, ILT3, ILT4, ILT5, and LIR8); (ii) activating, thosecontaining a short cytoplasmic tail and a charged amino acid residue inthe transmembrane domain (ILT1, ILT7, ILT8, and LIR6alpha) anddelivering an activating signal through the cytoplasmic immunoreceptortyrosine-based activating motif (ITAM) of the associated common gammachain of Fc receptor; and (iii) the soluble molecule ILT6 lacking thetransmembrane domain. A number of recent studies have highlightedimmunoregulatory roles for ILTs on the surface of antigen presentingcells (APC). ILT2, ILT3, and ILT4 receptors, the most characterizedimmune inhibitory receptors, are expressed on a wide range of immunecells including monocytes, B cells, dendritic cells, plasmacytoiddendritic cells and a subset of NK and T cells. ILT2 is expressed on Tcells subsets has been shown to inhibit activation and proliferation ofthese cells upon ligation (Colonna M. et al., J Immunol. 20011,66:2514-2521, J Immunol 2000; 165:3742-3755). ILT3 and ILT4 areupregulated by exposing immature DC to known immunosuppressive factors,including IL-10, vitamin D3, or suppressor CD8 T cells (Chang, C. C., etal., Nat Immunol, 3:237-243 (2002)). The expression of ILTs on DC istightly controlled by inflammatory stimuli, cytokines, and growthfactors, and is down-regulated following DC activation (Ju, X. S., etal., Gene, 331:159-164 (2004)). The expression of ILT2 and ILT4receptors is highly regulated by histone acetylation, which contributesto strictly controlled gene expression exclusively in the myeloidlineage of cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

Engagement of the inhibitory receptors ILT2 and ILT4 alters the cytokineand chemokine secretion/release profile of monocytes and can inhibit Fcreceptor signaling (Colonna, M., et al. J Leukoc Biol, 66:375-381(1999)). The role and function of ILT3 on DC have been preciselydescribed by the Suciu-Foca group (Suciu-Foca, N., Int Immunopharmacol,5:7-11 (2005)). Although the ligand for ILT3 is unknown, ILT4 is knownto bind to the third domain of HLA class I molecules (HLA-A, HLA-B,HLA-C, and HLA-G), competing with CD8 for MHC class I binding(Shiroishi, M., Proc Natl Acad Sci USA, 100:8856-8861 (2003)). Thepreferential ligand for several inhibitory ILT receptors is HLA-G. HLA-Gplays a potential role in maternal-fetal tolerance and in the mechanismsof escape of tumor cells from immune recognition and destruction (Hunt,J. S., et al., Faseb J, 19:681-693 (2005)). It is most likely thatregulation of DC function by HLA-G-ILT interactions is an importantpathway in the biology of DC. It has been determined that humanmonocyte-derived DC that highly express ILT2 and ILT4 receptors, whentreated with HLA-G and stimulated with allogeneic T cells, stillmaintain a stable tolerogenic-like phenotype (CD80low, CD86low,HLA-DRlow) with the potential to induce T cell anergy (Ristich, V., etal., Eur J Immunol, 35:1133-1142 (2005)). Moreover, the HLA-Ginteraction with DC that highly express ILT2 and ILT4 receptors resultedin down-regulation of several genes involved in the MHC class IIpresentation pathway. A lysosomal thiol reductase, IFN-gamma induciblelysosomal thiol reductase (GILT), abundantly expressed by professionalAPC, was greatly reduced in HLA-G-modified DC. The repertoire of primedCD4+ T cells can be influenced by DC expression of GILT, as in vivo Tcell responses to select antigens were reduced in animals lacking GILTafter targeted gene disruption (Marie, M., et al., Science,294:1361-1365 (2001)). The HLA-G/ILT interaction on DC interferes withthe assembly and transport of MHC class II molecules to the cellsurface, which might result in less efficient presentation or expressionof structurally abnormal MHC class II molecules. It was determined thatHLA-G markedly decreased the transcription of invariant chain (CD74),HLA-DMA, and HLA-DMB genes on human monocyte-derived DC highlyexpressing ILT inhibitory receptors (Ristich, V., et al; Eur J Immunol35:1133-1142 (2005)).

Another receptor of HLA-G is KIR2DL4 because KIR2DL4 binds to cellsexpressing HLA-G (US2003232051; Cantoni, C. et al. Eur J Immunol 28(1998) 1980; Rajagopalan, S. and E. O. Long. [published erratum appearsin J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093; Ponte, M. etal. PNAS USA 96 (1999) 5674). KIR2DL4 (also referred to as 2DL4) is a MRfamily member (also designated CD158d) that shares structural featureswith both activating and inhibitory receptors (Selvakumar, A. et al.Tissue Antigens 48 (1996) 285). 2DL4 has a cytoplasmic ITIM, suggestinginhibitory function, and a positively charged amino acid in thetransmembrane region, a feature typical of activating MR. Unlike otherclonally distributed KIRs, 2DL4 is transcribed by all NK cells(Valiante, N. M. et al. Immunity 7 (1997) 739; Cantoni, C. et al. Eur JImmunol 28 (1998) 1980; Rajagopalan, S. and E. O. Long. [publishederratum appears in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999)1093).

HLA-G has also been shown to interact with CD8 (Sanders et al, J. Exp.Med., 174 (1991), 737-740) on cytotoxic T cells and induce CD95 mediatedapoptosis in activated CD8 positive cytotoxic T cells (Fournel et al, J.Immun., 164 (2000), 6100-6104). This mechanism of elimination ofcytotoxic T cells has been reported to one of the mechanisms of immuneescape and induction of tolerance in pregnancy, inflammatory diseasesand cancer (Amodio G. et al, Tissue Antigens, 84 (2014), 255-263).

As used herein an anti-HLA-G antibody (either mono-, multi- orbispecific) or antigen binding moiety that “does not crossreact with” orthat “does not (specifically) bind to” a modified human HLA-G β2M MHC Icomplex, wherein the HLA-G specific amino acids have been replaced byHLA-A consensus amino acids, the complex comprising SEQ ID NO:40; amouse H2Kd β2M MHC I complex comprising SEQ ID NO:41 rat RT1A β2M MHC Icomplex comprising SEQ ID NO:43, human HLA-A2 β2M MHC I complexcomprising SEQ ID NO:35 and SEQ ID NO: 33 refers to an anti-HLA-Gantibody (either mono-, multi- or bispecific) or antigen binding moietythat does substantially not bind to any of these counterantigens. In oneembodiment an anti-HLA-G antibody(either mono-, multi- or bispecific) orantigen binding moiety that “does not crossreact with” or that “does notspecifically bind to” a modified human HLA-G β2M MHC I complex, whereinthe HLA-G specific amino acids have been replaced by HLA-A consensusamino acids, the complex comprising SEQ ID NO:40; a mouse H2Kd β2M MHC Icomplex comprising SEQ ID NO:41, a rat RT1A β2M MHC I complex comprisingSEQ ID NO:43, and/or a human HLA-A2 β2M MHC I complex comprising SEQ IDNO:35 and SEQ ID NO: 33 refers to an anti-HLA-G antibody (either mono-,multi- or bispecific) or antigen binding moiety that shows nosignificant binding/interaction in e.g. a Surface plasmon resonanceassay (as described e.g. in Example 2) The binding binding/interactionis determined with a standard binding assay, such as surface plasmonresonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden) with therespective antigen: a modified human HLA-G β2M MHC I complex, whereinthe HLA-G specific amino acids have been replaced by HLA-A consensusamino acids, the complex comprising SEQ ID NO:40; a mouse H2Kd β2M MHC Icomplex comprising SEQ ID NO:41 rat RT1A β2M MHC I complex comprisingSEQ ID NO:43, and/or a human HLA-A2 β2M MHC I complex comprising SEQ IDNO:35 and SEQ ID NO: 33 The assay setup as well as theconstruction/preparation of the antigens is described in the Examples.

The term “inhibits ILT2 binding to HLA-G on JEG-3 cells (ATCC HTB36)”refers to the inhibition of binding interaction of (recombinant) ILT2e.g in an assay as described in Example 5.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed on the surface of a T lymphocyte, particularly acytotoxic T lymphocyte, which is capable of inducing T cell activationupon interaction with an antibody. Specifically, interaction of anantibody with an activating T cell antigen may induce T cell activationby triggering the signaling cascade of the T cell receptor complex.

In a particular embodiment the activating T cell antigen is CD3,particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version189), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 88 for the human sequence;or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ IDNO: 108 for the cynomolgus [Macaca fascicularis] sequence).

“CD3” refers to any native CD3 from any vertebrate source, includingmammals such as primates (e.g. humans), non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. CD3 is an exemplary activated T cell antigen. The term “CD3”encompasses “full-length,” unprocessed CD3 as well as any form of CD3that results from processing in the cell. The term also encompassesnaturally occurring variants of CD3, e.g., splice variants or allelicvariants. In one embodiment, CD3 is human CD3, particularly the epsilonsubunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε isshown in UniProt (www.uniprot.org) accession no. P07766 (version 189),or NCBI (www.ncbi.nlm nih.gov/) RefSeq NP_000724.1. See also SEQ ID NO:88. The amino acid sequence of cynomolgus [Macaca fascicularis] CD3ε isshown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 89.

As used herein, an antibody (either mono-, multi- or bispecific) orantigen binding moiety “binding to human CD3”, “specifically binding tohuman CD3”, “that binds to human CD3” or “anti-CD3” refers to ananti-antibody (either mono-, multi- or bispecific) or antigen bindingmoiety specifically binding to the human CD3 antigen or itsextracellular domain (ECD) which shows significant binding/interactionin a surface plasmon resonance assay. In one embodiment with a bindingaffinity of a K_(D)-value of 5.0×10⁻⁸ mol/l or lower, in one embodimentof a K_(D)-value of 1.0×10⁻⁹ mol/l or lower, in one embodiment of aK_(D)-value of 5.0×10⁻⁸ mol/l to 1.0×10⁻¹³ mol/l. In one embodiment theantibody binds to CD3 comprising SEQ ID NO: 88.

The binding affinity is determined with a standard binding assay, suchas surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala,Sweden) e.g. using constructs comprising HLA-G extracellular domain(e.g. in its natural occurring 3 dimensional structure). In oneembodiment binding affinity is determined with a standard binding assayusing exemplary CD3 comprising SEQ ID NO: 88.

Accordingly a multispecific or bispecific that binds to human HLA-G andto human CD3, comprising a first antigen binding moiety that binds tohuman HLA-G and a second antigen binding moiety that binds to human CD3refers to an antibody that binds with an (first) antigen binding moietyto human HLA-G as described herein and that binds with another (second)antigen binding moiety to human CD3 as described herein.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. Suitable assays to measure T cell activation areknown in the art and described herein.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “bispecific” means that the antibody is able to specificallybind to at least two distinct antigenic determinants Typically, abispecific antibody comprises two antigen binding sites or moieties,each of which is specific for a different antigenic determinant. Incertain embodiments the bispecific antibody is capable of simultaneouslybinding two antigenic determinants, particularly two antigenicdeterminants expressed on two distinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antibody. As such, the term“monovalent binding to an antigen” denotes the presence of one (and notmore than one) antigen binding site specific for the antigen in theantibody.

The terms “antigen binding site” and “antigen binding moiety” as usedherein are interchangeable and refer to the site, i.e. one or more aminoacid residues, of an antibody which provides interaction with theantigen. For example, the antigen binding site of an antibody comprisesamino acid residues from the complement determining regions (CDRs). Anative immunoglobulin molecule typically has two antigen binding sites,a Fab molecule typically has a single antigen binding site. “Antigenbinding site” and “antigen binding moiety” refers to a polypeptidemolecule that specifically binds to an antigenic determinant. In oneembodiment, an antigen binding moiety is able to direct the entity towhich it is attached (e.g. a second antigen binding moiety) to a targetsite, for example to a specific type of tumor cell bearing the antigenicdeterminant In another embodiment an antigen binding moiety is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Antigen binding moieties include antibodiesand fragments thereof as further defined herein. Particular antigenbinding moieties include an antigen binding domain of an antibody,comprising an antibody heavy chain variable region and an antibody lightchain variable region. In certain embodiments, the antigen bindingmoieties may comprise antibody constant regions as further definedherein and known in the art. Useful heavy chain constant regions includeany of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constantregions include any of the two isotypes: κ and λ. In one preferredembodiment such constant regions are of human origin.

As used herein, the term “antigenic determinant” or “antigen” refers toa site on a polypeptide macromolecule to which an antigen binding moietybinds, forming an antigen binding moiety-antigen complex. Usefulantigenic determinants can be found, for example, on the surfaces oftumor cells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, on the surface of immune cells, free in bloodserum, and/or in the extracellular matrix (ECM).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively In one preferred embodiment such subclasses(isotypes) are of human origin. In one preferred embodiment theantibodies of the present invention are of the IgG isotype, in anotherpreferred embodiment of the IgG1 isotype

In one aspect, the antibody comprises a constant region of human origin.In one aspect, the antibody is an immunoglobulin molecule comprising ahuman constant region, particularly of the IgG isotype, moreparticularly of the IgG1 isotype, comprising a human CH1, CH2, CH3and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID Nos: 47 and 48 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 49 (human IgG1 heavy chain constant domainsCH1-CH2-CH3) or SEQ ID NO: 50 (human IgG1 heavy chain constant regionwith mutations L234A, L235A and P329G). An “effective amount” of anagent, e.g., a pharmaceutical formulation, refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including Fc domains (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one embodiment of theinvention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in an antibody or bispecific antibodyaccording to the invention, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment of the invention, a heavy chain including asubunit of an Fc domain as specified herein, comprised in an antibody orbispecific antibody according to the invention, comprises an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat). Compositions/formulations of the invention, such as thepharmaceutical compositions/formulations described herein, comprise apopulation of antibodies or bispecific antibodies of the invention. Thepopulation of antibodies or bispecific antibodies may comprise moleculeshaving a full-length heavy chain and molecules having a cleaved variantheavy chain. The population of antibodies or bispecific antibodies mayconsist of a mixture of molecules having a full-length heavy chain andmolecules having a cleaved variant heavy chain, wherein at least 50%, atleast 60%, at least 70%, at least 80% or at least 90% of the antibodiesor bispecific antibodies have a cleaved variant heavy chain. In oneembodiment of the invention a composition comprising a population ofantibodies or bispecific antibodies of the invention comprises anantibody or bispecific antibody comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). In one embodiment of the invention a compositioncomprising a population of antibodies or bispecific antibodies of theinvention comprises an antibody or bispecific antibody comprising aheavy chain including a subunit of an Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat). In one embodiment of the invention such acomposition comprises a population of antibodies or bispecificantibodies comprised of molecules comprising a heavy chain including asubunit of an Fc domain as specified herein; molecules comprising aheavy chain including a subunit of a Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat); and molecules comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain. In onepreferred embodiment such an Fc domain is of human origin, in onepreferred of the IgG isotype, in another preferred embodiment of theIgG1 isotype.

“Framework” or “FR” refers to variable domain residues other thancomplement determining region (CDR) residues. The FR of a variabledomain generally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the CDR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human CDRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “complementarity determining regions” or “CDRs” as used hereinrefers to each of the regions of an antibody variable domain which arehypervariable in sequence and/or form structurally defined loops(“hypervariable loops”) and/or contain the antigen-contacting residues(“antigen contacts”). Generally, antibodies comprise six CDRs: three inthe VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2,CDR-L3). Exemplary CDRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55        (CDR-H2), and 96-101 (CDR-H3) (Chothia and Lesk, J. Mol. Biol.        196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (CDR-L1), 50-56        (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and        95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of        Immunological Interest, 5th Ed. Public Health Service, National        Institutes of Health, Bethesda, Md. (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58        (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol.        262: 732-745 (1996)); and    -   (d) combinations of (a), (b), and/or (c), including CDR amino        acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (vL3), 31-35        (CDR-H1), 50-63 (CDR-H2), and 95-102 (CDR-H3).

Unless otherwise indicated, CDR-residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In one embodiment the antibody is anisolated antibody. In some embodiments, an antibody is purified togreater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-HLA-G antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain. Such modification promoting the associationof the first and the second subunit of the Fc domain play an importantrole in the heterodimerization of multi-or bispecific antibodies (seee.g. also below under A.2 Exemplary multispecific anti-HLA-G/anti-CD3Antibodies)

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three complement determining regions (CDRs). (See,e.g., Kindt, T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman andCo., N.Y. (2007), page 91) A single VH or VL domain may be sufficient toconfer antigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See e.g., Portolano, S. et al., J. Immunol.150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

I. Compositions and Methods

In one aspect, the invention is based, in part, on the finding thatsurprisingly among various variants of HLA-G-0090 in which theglcyosylation site was removed only the two variants HLA-G-0090-VL-S32Pand HLA-G-0090-VL-S33A show even improved binding properties, goodexpressability and stability, while showing no more glycosylation at theCDR-L1 of the LC (so no Fab glycosylation could be detected). As allrecently approved pharmaceutical antibody products are produced inmammalian cells, especially CHO cells (see e.g. Walsh G., Nature Biotech(2018) 1136-1145) providing an antibody without glycosylation sites inthe binding region (VH and VL and especially the CDRs) represents avaluable advantage, as these antibodies can then be readily used forproduction in mammalian expression systems without the risk of (at leastpartially impairing the binding properties by glycosylation. Inparticular for Fc domain-comprising antibodies, manufacturing theantibody in a host cell lacking a glycosylation machinery (such as aprokaryotic host cell) would not results in a product of comparablequality, since the N-glycans attached to amino acid residue ASN297(numbering according to EU index of Kabat) in the Fc region are requiredto maintain solubility and thermal stability, and to prevent aggregationof the antibody in an aqueous solution (e.g. in pharmaceuticalcompositions).

A.1 Exemplary Anti-HLA-G Antibodies

One embodiment of the invention is an antibody that binds to human HLA-Gcomprising

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:24.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:24.

In embodiment such anti-HLA-G antibody has improved binding propertieswith respect to maximal binding (Rmax) and/or binding affinity (KD)compared to the (parental) antibody that comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising the amino acid sequence of SEQ ID NO:8 (as shown in Example2). In a further embodiment the HLA-G antibody of the present inventioncomprises an Fc domain of human origin, in one embodiment the Fc domainis of the IgG isotype, in one preferred embodiment of the IgG1 isotype.In one embodiment, such an IgG1 isotype Fc domain of human origincomprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”,“PGLALA” or “LALAPG”) (numberings according to Kabat EU index).

In a further embodiment the HLA-G antibody of the present inventioncomprises a constant region of human origin, particularly of the IgGisotype, more particularly of the IgG1 isotype, comprising a human CH1,CH2, CH3 and/or CL domain.

In one embodiment such such constant region of the IgG1 isotypecomprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”,“PGLALA” or “LALAPG”) (numberings according to Kabat EU index).

Such Fc domain-comprising antibodies can be typically N-glycosylated atposition ASN-297 (numbering according to Kabat EU index) e.g. whenproduced in eukaryotic cells, like mammalian cells, in particular CHOcells. N-glycosylation at position ASN-297 (numbering according to EUindex (see Kabat) represents a valuable contribution to e.g. the highstability, low aggregation tendency and/or good pharmacokinetic andother critical quality properties of such an antibody (see e.g. Zheng etal, mAbs (2011) 568-576; and Reusch et al, Glycobiology (2015)1325-133). Therefore such an Fc domain-comprising antibody of thepresent invention is easily ready for production in in eukaryotic cells,like mammalian cells, in particular CHO cells, without the risk of beingglycosylated in the binding region (which would interfere with itsbinding properties), but at the same time with the benefit and valuablequality attributes of the N-glycosylation at position ASN-297.

One embodiment of the invention is an antibody that binds to human HLA-Gwherein the antibody comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:24, wherein antibody has improved bindingproperties with respect to maximal binding (Rmax) and/or bindingaffinity (KD) compared to the (parental) antibody that comprises a VHdomain comprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising the amino acid sequence of SEQ ID NO:8 (as shown in Example2).

In Embodiment the Anti-HLA-G Antibody

a) does not crossreact with a modified human HLA-G β2M MHC I complex,wherein the HLA-G specific amino acids have been replaced by HLA-Aconsensus amino acids, the complex comprising SEQ ID NO:40; and/or

b) does not crossreact with a mouse H2Kd β2M MHC I complex comprisingSEQ ID NO:41; and/or

c) does not crossreact with rat RT1Aβ2M MHC I complex comprising SEQ IDNO:43.

In Embodiment the Anti-HLA-G Antibody

a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells (ATCC No.HTB36); or

b) binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36), andinhibits ILT2 binding to (HLA-G expressed on) JEG-3 cells (ATCC No.HTB36).

In another aspect, the invention relates to multispecific antibodiescomprising the anti-HLA-G antigen binding moiety. These multispecificantibodies (e.g. the bispecific antibodies) as described herein use theselected, improved anti-HLA-G antibodies as first antigen bindingmoiety/site. These anti-HLA-G antibodies bind to toHLA-G with highspecificity and affinity (improved binding properties, nocrossreactivity with other species and human HLA-A consensus sequences),and have ability to specifically inhibit ILT2 and or ILT4 binding toHLA-G.

In one embodiment the invention relates to a multispecific (preferablybispecific) that binds to human HLA-G and to human CD3. In oneembodiment the invention relates to a multispecific (preferablybispecific) anti-HLA-G/anti-CD3 antibody, wherein the multispecific(preferably bispecific) antibody that binds to human HLA-G and to humanCD3, comprises a first antigen binding moiety that binds to human HLA-Gand a second antigen binding moiety that binds to human CD3. Thisbispecific antibody as described herein binds with specific, secondantigen binding moieties/sites to CD3, especially CD3epsiln and aretherefore able to attract CD3 expressing T-cells to HLA-G expressingtumor cells and at the same time to inhibit the HLA-G induced immunesuppression in the tumor environment by blocking ILT2/4 binding toHLA-G. Thus these bispecific anti-HLA-G/anti-CD3 antibodies show strongtumor growth inhibition and tumor regression in vivo.

A.2 Exemplary Multispecific Anti-HLA-G/Anti-CD3 Antibodies

One embodiment of the invention is a bispecific antibody that binds tohuman HLA-G and to human CD3, comprising a first antigen binding moietythat binds to human HLA-G and a second antigen binding moiety that bindsto human CD3, wherein the first antigen binding moiety that binds tohuman HLA-G comprises

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6;

and wherein the second antigen binding moiety that binds to a T cellactivating antigen binds to human CD3 comprises

C) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:52, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:53, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:54; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:55; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:56 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:57, or

C) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:60, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:61, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:62; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:63; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:64 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:65, or

D) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:68, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:69, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:70; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:71; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:72 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:73.

-   -   Another embodiment of the invention is a bispecific antibody        that binds to human HLA-G and to human CD3, comprising a first        antigen binding moiety that binds to human HLA-G and a second        antigen binding moiety that binds to human CD3,

wherein the first antigen binding moiety

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26,

and wherein the second antigen binding moiety

-   -   C) comprises a VH domain comprising the amino acid sequence of        SEQ ID NO:58 and a VL domain comprising the amino acid sequence        of SEQ ID NO:59; or

D) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:66 and a VL domain comprising the amino acid sequence of SEQ IDNO:67; or

E) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:74 and a VL domain comprising the amino acid sequence of SEQ IDNO:75.

-   -   Another embodiment of the invention is a bispecific antibody        that binds to human HLA-G and to human CD3, comprising a first        antigen binding moiety that binds to human HLA-G and a second        antigen binding moiety that binds to human CD3,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

-   -   comprises a VH domain comprising the amino acid sequence of SEQ        ID NO:58 and a VL domain comprising the amino acid sequence of        SEQ ID NO:59.    -   Another embodiment of the invention is a bispecific antibody        that binds to human HLA-G and to human CD3, comprising a first        antigen binding moiety that binds to human HLA-G and a second        antigen binding moiety that binds to human CD3,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

-   -   comprises a VH domain comprising the amino acid sequence of SEQ        ID NO:66 and a VL domain comprising the amino acid sequence of        SEQ ID NO:67.    -   Another embodiment of the invention is a bispecific antibody        that binds to human HLA-G and to human CD3, comprising a first        antigen binding moiety that binds to human HLA-G and a second        antigen binding moiety that binds to human CD3,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:74and a VL domain comprising the amino acid sequence of SEQ ID NO:75.

-   -   In one embodiment these bispecific antibodies are characterized        by one or more of the following properties:

a) induction of T cell mediated cytotoxicity/tumor cell killing in thepresence of HLA-G expressing tumor cells (preferably in the presence ofJEG3 cells (ATCC No. HTB36)); and/or

b) induction IFN gamma secretion by T cells in the presence of HLA-Gexpressing tumor cells (preferably in the presence of JEG3 cells (ATCCNo. HTB36)); and/or

c) inhibition of tumor growth in vivo (in a mouse xenograft tumormodel),

-   -   d) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example        13); and/or

e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (seeExample 14).

In one embodiment these bispecific antibodies are characterized inaddition by one or more of the following properties: the bispecificantibody

a) does not crossreact with a modified human HLA-G β2M MHC I complex,wherein the HLA-G specific amino acids have been replaced by HLA-Aconsensus amino acids, the complex comprising SEQ ID NO:44; and/or

b) does not crossreact with a mouse H2Kd β2M MHC I complex comprisingSEQ ID NO:41; and/or

c) does not crossreact with rat RT1A β2M MHC I complex comprising SEQ IDNO:43.

-   -   In one embodiment these bispecific antibodies are characterized        in addition by one or more of the following properties: the        bispecific antibody

a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells (ATCC No.HTB36); or

b) binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36), andinhibits ILT2 binding to (HLA-G expressed on) JEG-3 cells (ATCC No.HTB36); and/or

Multispecific Antibodies

Multispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different sites, i.e., different epitopeson different antigens or different epitopes on the same antigen. Incertain embodiments, the multispecific antibody has three or morebinding specificities. In a preferred embodiment the multispecificantibody provided herein is a bispecific antibody. In certainembodiments, one of the binding specificities is for HLA-G and the otherspecificity is for CD3. In certain embodiments, bispecific antibodiesmay bind to two (or more) different epitopes of HLA-G. Multispecificantibodies can be prepared as full length antibodies or antibodyfragments.

Techniques for making multispecific and in particular bispecificantibodies include, but are not limited to, recombinant co-expression oftwo immunoglobulin heavy chain-light chain pairs having differentspecificities (see Milstein and Cuello, Nature 305: 537 (1983)) and“knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, andAtwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodiesmay also be made by engineering electrostatic steering effects formaking antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelnyet al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); usingthe common light chain technology for circumventing the light chainmis-pairing problem (see, e.g., WO 98/50431); using “diabody” technologyfor making bispecific antibody fragments (see, e.g., Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chainFv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994));and preparing trispecific antibodies as described, e.g., in Tutt et al.J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites,including for example, “Octopus antibodies,” or DVD-Ig are also includedherein (see, e.g. WO 2001/77342 and WO 2008/024715). Other examples ofmultispecific antibodies with three or more antigen binding sites can befound in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792,and WO 2013/026831. The bispecific antibody or antigen binding fragmentthereof also includes a “Dual Acting FAb” or “DAF” comprising an antigenbinding site that binds to HLA-G as well as another different antigen,or two different epitopes of HLA-G (see, e.g., US 2008/0069820 and WO2015/095539).

Multi-specific antibodies may also be provided in an asymmetric formwith a domain crossover in one or more binding arms of the same antigenspecificity, i.e. by exchanging the VH/VL domains (see e.g., WO2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, andKlein at al., MAbs 8 (2016) 1010-20). Asymmetrical Fab arms can also beengineered by introducing charged or non-charged amino acid mutationsinto domain interfaces to direct correct Fab pairing. See e.g., WO2016/172485.

Various further molecular formats for multispecific antibodies are knownin the art and are included herein (see e.g., Spiess et al., Mol Immunol67 (2015) 95-106).

A particular type of multispecific antibodies, also included herein, arebispecific antibodies designed to simultaneously bind to a surfaceantigen on a target cell, e.g., a tumor cell, and to an activating,invariant component of the T cell receptor (TCR) complex, such as CD3,for retargeting of T cells to kill target cells. Hence, in certainembodiments, an antibody provided herein is a multispecific antibody,particularly a bispecific antibody, wherein one of the bindingspecificities is for HLA-G and the other is for CD3.

Examples of bispecific antibody formats that may be useful for thispurpose include, but are not limited to, the so-called “BiTE”(bispecific T cell engager) molecules wherein two scFv molecules arefused by a flexible linker (see, e.g., WO2004/106381, WO2005/061547,WO2007/042261, and WO2008/119567, Nagorsen and Bäuerle, Exp Cell Res317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305(1996)) and derivatives thereof, such as tandem diabodies (“TandAb”;Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinityretargeting) molecules which are based on the diabody format but featurea C-terminal disulfide bridge for additional stabilization (Johnson etal., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which arewhole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., CancerTreat Rev 36, 458-467 (2010)). Particular T cell bispecific antibodyformats included herein are described in WO 2013/026833, WO2013/026839,WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

Bispecific Antibodies that Bind to HLA-G and to CD3

The invention also provides a bispecific antibody, i.e. an antibody thatcomprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants (a first and a secondantigen).

Based on the anti-HLA-G antigen binding moieties and anti-CD3 antigenbinding moieties they developed, the present inventors have developedbispecific antibodies that bind to HLA-G and to CD3.

As shown in the Examples, these bispecific antibodies have a number ofremarkable properties, including good efficacy and low toxicity.

Thus, in certain aspects, the invention provides a bispecific antibody,comprising (a) a first antigen binding moiety that binds to human HLA-G,and (b) a second antigen binding moiety which specifically binds tohuman CD3, wherein the bispecific antibody has any of the followingfeatures:-The bispecific antibody of the invention specifically inducesT-cell mediated killing of cells expressing HLA-G. In some embodiments,the bispecific antibody of the invention specifically induces T-cellmediated killing of cells expressing HLA-G. In a more specificembodiment, the bispecific antibody specifically induces T-cell mediatedkilling of cells expressing HLA-G.

In one embodiment, induction of T-cell mediated killing by thebispecific antibody is determined using HLA-G -expressing cells.

In one embodiment, activation of T cells by the bispecific antibody isdetermined by measuring, particularly by flow cytometry, expression ofCD25 and/or CD69 by T cells after incubation with the bispecificantibody in the presence of HLA-G-expressing cells.

In a specific embodiment, induction of T-cell mediated killing by thebispecific antibody is determined as follows:

Ability of anti HLA-G/anti CD3 TCB to activate T cells in the presenceof HLA-G expressing tumor cells is tested on SKOV3 cells transfectedwith recombinant HLA-G (SKOV3HLA-G). Activation of T cells is assessedby FACS analysis of cell surface activation markers CD25 and earlyactivation marker CD69 on T cells. Briefly, Peripheral Blood MononuclearCells (PBMCs) are isolated from human peripheral blood by densitygradient centrifugation using Lymphocyte Separating Medium Tubes (PAN#P04-60125). PBMC's and SKOV3HLA-G cells are seeded at a ratio of 10:1in 96-well U bottom plates. The co-culture is then incubated withHLA-G-TCB at different concentrations as described in the Example 12 andincubated for 24 h at 37° C. in an incubator with 5% Co2. On the nextday, expression of CD25 and CD69 is measured by flow cytometry.

For flow cytometry analysis, cells are stained with with PerCP-Cy5.5Mouse Anti-Human CD8 (BD Pharmingen #565310), PE Mouse Anti-Human CD25(eBioscience #9012-0257) and APC Mouse Anti-Human CD69 (BD Pharmingen#555533) at 4° C. Briefly, antibodies are diluted to a 2-foldconcentration and 25 μl of antibody dilution are added in each well with25 μl of pre-washed co-cultures. Cells are stained for 30 min at 4° C.and washed twice with 200 μl well staining buffer and centrifugation at300 g for 5 min. Cell pellets are resuspended in 200 μl of stainingbuffer and stained with DAPI for live dead discrimination at a finalconcentration of 2 μg/ml. Samples are then measured using BD LSR flowcytometer. Data analysis is performed using FlowJo V.10.1 software.

The bispecific antibody of the invention specifically activates T cellsin the presence of cells expressing HLA-G. In some embodiments, thebispecific antibody of the invention specifically activates T cells inthe presence of cells expressing HLA-G. In a more specific embodiment,the bispecific antibody specifically activates T cells in the presenceof cells expressing HLA-G.

In one embodiment, the bispecific antibody induces T cell mediatedkilling of, or activate T cells in the presence of, cells expressingHLA-G. In one embodiment, the bispecific antibody induces T cellmediated killing of, and/or activates T cells in the presence of, cellsexpressing HLA-G with an EC50 that is at least 5, at least 10, at least15, at least 20, at least 25, at least 50, at least 75 or at least 100times lower than the EC50 for induction of T cell mediated killing of,or activation of T cells in the presence of, cells expressing HLA-G

According to particular embodiments of the invention, the antigenbinding moieties comprised in the bispecific antibody are Fab molecules(i.e. antigen binding domains composed of a heavy and a light chain,each comprising a variable and a constant domain). In one embodiment,the first and/or the second antigen binding moiety is a Fab molecule. Inone embodiment, said Fab molecule is human. In a particular embodiment,said Fab molecule is humanized In yet another embodiment, said Fabmolecule comprises human heavy and light chain constant domains.

Preferably, at least one of the antigen binding moieties is a crossoverFab molecule. Such modification reduces mispairing of heavy and lightchains from different Fab molecules, thereby improving the yield andpurity of the bispecific antibody of the invention in recombinantproduction. In a particular crossover Fab molecule useful for thebispecific antibody of the invention, the variable domains of the Fablight chain and the Fab heavy chain (VL and VH, respectively) areexchanged. Even with this domain exchange, however, the preparation ofthe bispecific antibody may comprise certain side products due to aso-called Bence Jones-type interaction between mispaired heavy and lightchains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To furtherreduce mispairing of heavy and light chains from different Fab moleculesand thus increase the purity and yield of the desired bispecificantibody, charged amino acids with opposite charges may be introduced atspecific amino acid positions in the CH1 and CL domains of either theFab molecule(s) binding to the first antigen (HLA-G), or the Fabmolecule binding to the second antigen an activating T cell antigen suchas CD3, as further described herein. Charge modifications are madeeither in the conventional Fab molecule(s) comprised in the bispecificantibody (such as shown e.g. in FIGS. 13A-C, G-J), or in the VH/VLcrossover Fab molecule(s) comprised in the bispecific antibody (such asshown e.g. in FIGS. 13D-F, K-N) (but not in both). In particularembodiments, the charge modifications are made in the conventional Fabmolecule(s) comprised in the bispecific antibody (which in particularembodiments bind(s) to the first antigen, i.e. HLA-G).

In a particular embodiment according to the invention, the bispecificantibody is capable of simultaneous binding to the first antigen (i.e.HLA-G), and the second antigen (e.g. an activating T cell antigen,particularly CD3). In one embodiment, the bispecific antibody is capableof crosslinking a T cell and a target cell by simultaneous binding HLA-Gand an activating T cell antigen. In an even more particular embodiment,such simultaneous binding results in lysis of the target cell,particularly a HLA-G expressing tumor cell. In one embodiment, suchsimultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the bispecificantibody to the activating T cell antigen, particularly CD3, withoutsimultaneous binding to HLA-G does not result in T cell activation.

In one embodiment, the bispecific antibody is capable of re-directingcytotoxic activity of a T cell to a target cell. In a particularembodiment, said re-direction is independent of MHC-mediated peptideantigen presentation by the target cell and and/or specificity of the Tcell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

First Antigen Binding Moiety that Binds to Human HLA-G

The bispecific antibody of the invention comprises at least one antigenbinding moiety, particularly a Fab molecule, that binds to human HLA-G(first antigen). In certain embodiments, the bispecific antibodycomprises two antigen binding moieties, particularly Fab molecules,which bind to human HLA-G. In a particular such embodiment, each ofthese antigen binding moieties binds to the same antigenic determinant.In an even more particular embodiment, all of these antigen bindingmoieties are identical, i.e. they comprise the same amino acid sequencesincluding the same amino acid substitutions in the CH1 and CL domain asdescribed herein (if any). In one embodiment, the bispecific antibodycomprises not more than two antigen binding moieties, particularly Fabmolecules, which bind to human HLA-G.

In particular embodiments, the antigen binding moiety(ies) which bind tohuman HLA-G is/are a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to a second antigen is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CH1 and CL of theFab heavy and light chains are exchanged/replaced by each other.

In alternative embodiments, the antigen binding moiety(ies)which bind tohuman HLA-G is/are a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In such embodiments, the antigenbinding moiety(ies) that binds a second antigen is a conventional Fabmolecule.

The HLA-G binding moiety is able to direct the bispecific antibody to atarget site, for example to a specific type of tumor cell that expresseshuman HLA-G.

The first antigen binding moiety of the bispecific antibody mayincorporate any of the features, singly or in combination, describedherein in relation to the antibody that binds HLA-G, unlessscientifically clearly unreasonable or impossible.

Thus, in one aspect, the invention provides a bispecific antibody,comprising a first antigen binding moiety that binds to a first antigen,wherein the first antigen is human HLA-G (in one embodiment the antibodybinds to HLA-G β2M MHC I complex comprising SEQ ID NO: 39), and thefirst antigen binding moiety comprises (a) a VH domain comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3comprising an amino acid sequence of SEQ ID NO:3; and wherein the VHdomain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,99% or 100% (in one preferred embodiment 98% or 99% or 100%) sequenceidentity to the amino acid sequence of SEQ ID NO: 7; and (b) a VL domaincomprising (i) CDR-L1 comprising the amino acid sequence of SEQ IDNO:23; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and(iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; andwherein the VL domain comprises an amino acid sequence of at least 95%,96%, 97%, 98%, 99% or 100% (in one preferred embodiment 98% or 99% or100%) sequence identity to the amino acid sequence of SEQ ID NO: 24.

The term like “a VH domain comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:2, and (iii) CDR-H3 comprising an amino acidsequence of SEQ ID NO:3; and wherein the VH domain comprises an aminoacid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in onepreferred embodiment 98% or 99% or 100%) sequence identity to the aminoacid sequence of SEQ ID NO: 7” refers to a VH domain with an amino acidsequence of SEQ ID NO: 7 wherein the 3 CDRs are unchanged (i.e. the sameas in SEQ ID NO:7) but e.g. no, one, two, three, four or five amino acidresidues in the framework regions of the VH is/are changed/substitutedwith another amino acid without affecting the binding properties of theVH and the antigen binding site. As the framework residues with a highprobability to influence on the binding properties are well known (seee.g. Foote J. and Winter G., J. Mol. Biol. (1992) 224, 487-499), theframework residues with no or minor influence can be chosen forsubstitution. The same applies to analogues terms used herein relatingto another VH or VL.

-   -   In one embodiment the first antigen binding moiety comprises a        VH domain comprising the amino acid sequence of SEQ ID NO:7 and        a VL domain comprising the amino acid sequence of SEQ ID NO:24.

In one embodiment the first binding moiety that binds to human HLA-G (inone embodiment to HLA-G β2M MHC I complex comprising SEQ ID NO: 39),comprises

-   -   (a) a VH domain comprising (i) CDR-H1 comprising the amino acid        sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid        sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising an amino        acid sequence of SEQ ID NO:3; and wherein the VH domain        comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,        99% or 100% (in one preferred embodiment 98% or 99% or 100%)        sequence identity to the amino acid sequence of SEQ ID NO: 7;        and (b) a VL domain comprising (i) CDR-L1 comprising the amino        acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the amino        acid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the        amino acid sequence of SEQ ID NO:6; and wherein the VL domain        comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,        99% or 100% (in one preferred embodiment 98% or 99% or 100%)        sequence identity to the amino acid sequence of SEQ ID NO: 26.    -   In one embodiment the first antigen binding moiety comprises a        VH domain comprising the amino acid sequence of SEQ ID NO:7 and        a VL domain comprising the amino acid sequence of SEQ ID NO:26.

Such anti-HLA-G antibodies show highly valuable properties, as they haveno N-glcyosylation in the antigen binding site (and the CDR-L1) (asshown in Example 2), have improved binding properties with respect tomaximal binding (Rmax) and/or binding affinity (KD) compared to the(parental) antibody that comprises a VH domain comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising the amino acidsequence of SEQ ID NO:8 (as shown in Example 2), do not crossreact witha HLA-A MHC I complexes, or murine or rat MHC I complexes and bind to(HLA-G expressed on) JEG3 cells (ATCC No. HTB36), and inhibits ILT2binding to (HLA-G expressed on) JEG-3 cells (ATCC No. HTB36).

Second Antigen Binding Moiety that Binds to Human CD3

The bispecific antibody of the invention comprises at least one antigenbinding moiety, particularly a Fab molecule, that binds to human CD3.

In particular embodiments, the antigen binding moiety that binds humanCD3, is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL or the constant domainsCH1 and CL of the Fab heavy and light chains are exchanged/replaced byeach other. In such embodiments, the antigen binding moiety(ies) thatbinds to human HLA-G is preferably a conventional Fab molecule. Inembodiments where there is more than one antigen binding moiety,particularly Fab molecule, that binds to human CD3 comprised in thebispecific antibody, the antigen binding moiety that binds human CD3preferably is a crossover Fab molecule and the antigen binding moietiesthat bind to human HLA-G are conventional Fab molecules.

In alternative embodiments, the antigen binding moiety that binds to thesecond antigen is a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to the first antigen (i.e. HLA-G)is a crossover Fab molecule as described herein, i.e. a Fab moleculewherein the variable domains VH and VL or the constant domains CH1 andCL of the Fab heavy and light chains are exchanged/replaced by eachother. In embodiments where there is more than one antigen bindingmoiety, particularly Fab molecule, that binds to a second antigencomprised in the bispecific antibody, the antigen binding moiety thatbinds to human HLA-G preferably is a crossover Fab molecule and theantigen binding moieties that bind to human CD3 are conventional Fabmolecules.

In some embodiments, the second antigen is an activating T cell antigen(also referred to herein as an “activating T cell antigen bindingmoiety, or activating T cell antigen binding Fab molecule”). In aparticular embodiment, the bispecific antibody comprises not more thanone antigen binding moiety capable of specific binding to an activatingT cell antigen. In one embodiment the bispecific antibody providesmonovalent binding to the activating T cell antigen.

In particular embodiments, the second antigen is CD3, particularly humanCD3 (SEQ ID NO: 88) or cynomolgus CD3 (SEQ ID NO: 89), most particularlyhuman CD3. In one embodiment the second antigen binding moiety iscross-reactive for (i.e. specifically binds to) human and cynomolgusCD3. In some embodiments, the second antigen is the epsilon subunit ofCD3 (CD3 epsilon).

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises (a) a VH domain comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:52, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:53, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:54; and wherein the VH domain comprises an aminoacid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in onepreferred embodiment 98% or 99% or 100%) sequence identity to the aminoacid sequence of SEQ ID NO: 58; and (b) a VL domain comprising (i)CDR-L1 comprising the amino acid sequence of SEQ ID NO:55; (ii) CDR-L2comprising the amino acid sequence of SEQ ID NO:56 and (iii) CDR-L3comprising the amino acid sequence of SEQ ID NO:57, and wherein the VLdomain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,99% or 100% (in one preferred embodiment 98% or 99% or 100%) sequenceidentity to the amino acid sequence of SEQ ID NO: 59.

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises (a) a VH domain comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:60, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:61, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:62, and wherein the VH domain comprises an aminoacid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in onepreferred embodiment 98% or 99% or 100%) sequence identity to the aminoacid sequence of SEQ ID NO: 66; and (b) a VL domain comprising (i)CDR-L1 comprising the amino acid sequence of SEQ ID NO:63; (ii) CDR-L2comprising the amino acid sequence of SEQ ID NO:64 and (iii) CDR-L3comprising the amino acid sequence of SEQ ID NO:65, and wherein the VLdomain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,99% or 100% (in one preferred embodiment 98% or 99% or 100%) sequenceidentity to the amino acid sequence of SEQ ID NO: 67.

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises (a) a VH domain comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:68, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:69, and wherein the VH domain comprises an aminoacid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in onepreferred embodiment 98% or 99% or 100%) sequence identity to the aminoacid sequence of SEQ ID NO: 74; and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:70; and (b) a VL domain comprising (i) CDR-L1comprising the amino acid sequence of SEQ ID NO:71; (ii) CDR-L2comprising the amino acid sequence of SEQ ID NO:72 and (iii) CDR-L3comprising the amino acid sequence of SEQ ID NO:73, and wherein the VLdomain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,99% or 100% (in one preferred embodiment 98% or 99% or 100%) sequenceidentity to the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises a VH domain comprising the amino acid sequence of SEQ IDNO: 58, and a VL domain comprising the amino acid sequence of SEQ ID NO:59.

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises a VH domain comprising the amino acid sequence of SEQ IDNO: 66, and a VL domain comprising the amino acid sequence of SEQ ID NO:67.

In one embodiment, the second antigen binding moiety that binds to humanCD3 comprises a VH domain comprising the amino acid sequence of SEQ IDNO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO:75.

Such CD3 antigen binding moietiys/sites show highly valuable properties(e.g. when provided as bispecific antibodies binding to CD3 and HLA-G(with the HLA-G antigen binding moieties as described herein). Theyshow{circumflex over ( )}

a) good thermal stability

b) induction IFN gamma secretion by T cells in the presence of HLA-Gexpressing tumor cells (preferably in the presence of JEG3 cells (ATCCNo. HTB36)) (Example 11); and/or

c) induction of T cell mediated cytotoxicity/tumor cell killing in thepresence of HLA-G expressing tumor cells (preferably in the presence ofJEG3 cells (ATCC No. HTB36)) (Example 12); and/or

d) inhibition of tumor growth in vivo (in a mouse xenograft tumormodel),

-   -   e) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example        13); and/or

f) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (seeExample 14).

In some embodiments, the second antigen binding moiety is a Fab moleculewherein the variable domains VL and VH or the constant domains CL andCH1, particularly the variable domains VL and VH, of the Fab light chainand the Fab heavy chain are replaced by each other (i.e. according tosuch embodiment, the second antigen binding moiety is a crossover Fabmolecule wherein the variable or constant domains of the Fab light chainand the Fab heavy chain are exchanged). In one such embodiment, thefirst (and the third, if any) antigen binding moiety is a conventionalFab molecule.

In one embodiment, not more than one antigen binding moiety that bindsto the second antigen (e.g. an activating T cell antigen such as CD3) ispresent in the bispecific antibody (i.e. the bispecific antibodyprovides monovalent binding to the second antigen).

Charge Modifications

The bispecific antibodies of the invention may comprise amino acidsubstitutions in Fab molecules comprised therein which are particularlyefficient in reducing mispairing of light chains with non-matching heavychains (Bence-Jones-type side products), which can occur in theproduction of Fab-based bi-/antibodies with a VH/VL exchange in one (ormore, in case of molecules comprising more than two antigen-binding Fabmolecules) of their binding arms (see also PCT publication no. WO2015/150447, particularly the examples therein, incorporated herein byreference in its entirety). The ratio of a desired bispecific antibodycompared to undesired side products, in particular Bence Jones-type sideproducts occurring in bispecific antibodies with a VH/VL domain exchangein one of their binding arms, can be improved by the introduction ofcharged amino acids with opposite charges at specific amino acidpositions in the CH1 and CL domains (sometimes referred to herein as“charge modifications”).

Accordingly, in some embodiments wherein the first and the secondantigen binding moiety of the bispecific antibody are both Fabmolecules, and in one of the antigen binding moieties (particularly thesecond antigen binding moiety) the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,

-   -   i) in the constant domain CL of the first antigen binding moiety        the amino acid at position 124 is substituted by a positively        charged amino acid (numbering according to Kabat), and wherein        in the constant domain CH1 of the first antigen binding moiety        the amino acid at position 147 or the amino acid at position 213        is substituted by a negatively charged amino acid (numbering        according to Kabat EU index); or    -   ii) in the constant domain CL of the second antigen binding        moiety the amino acid at position 124 is substituted by a        positively charged amino acid (numbering according to Kabat),        and wherein in the constant domain CH1 of the second antigen        binding moiety the amino acid at position 147 or the amino acid        at position 213 is substituted by a negatively charged amino        acid (numbering according to Kabat EU index).    -   The bispecific antibody does not comprise both modifications        mentioned under i) and ii). The constant domains CL and CH1 of        the antigen binding moiety having the VH/VL exchange are not        replaced by each other (i.e. remain unexchanged).

In a more specific embodiment,

-   -   i) in the constant domain CL of the first antigen binding moiety        the amino acid at position 124 is substituted independently by        lysine (K), arginine (R) or histidine (H) (numbering according        to Kabat), and in the constant domain CH1 of the first antigen        binding moiety the amino acid at position 147 or the amino acid        at position 213 is substituted independently by glutamic acid        (E), or aspartic acid (D) (numbering according to Kabat EU        index); or    -   ii) in the constant domain CL of the second antigen binding        moiety the amino acid at position 124 is substituted        independently by lysine (K), arginine (R) or histidine (H)        (numbering according to Kabat), and in the constant domain CH1        of the second antigen binding moiety the amino acid at position        147 or the amino acid at position 213 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index).

In one such embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

-   -   In a further embodiment, in the constant domain CL of the first        antigen binding moiety the amino acid at position 124 is        substituted independently by lysine (K), arginine (R) or        histidine (H) (numbering according to Kabat), and in the        constant domain CH1 of the first antigen binding moiety the        amino acid at position 147 is substituted independently by        glutamic acid (E), or aspartic acid (D) (numbering according to        Kabat EU index).    -   In a particular embodiment, in the constant domain CL of the        first antigen binding moiety the amino acid at position 124 is        substituted independently by lysine (K), arginine (R) or        histidine (H) (numbering according to Kabat) and the amino acid        at position 123 is substituted independently by lysine (K),        arginine (R) or histidine (H) (numbering according to Kabat),        and in the constant domain CH1 of the first antigen binding        moiety the amino acid at position 147 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index) and the amino acid at        position 213 is substituted independently by glutamic acid (E),        or aspartic acid (D) (numbering according to Kabat EU index).    -   In a more particular embodiment, in the constant domain CL of        the first antigen binding moiety the amino acid at position 124        is substituted by lysine (K) (numbering according to Kabat) and        the amino acid at position 123 is substituted by lysine (K)        (numbering according to Kabat), and in the constant domain CH1        of the first antigen binding moiety the amino acid at position        147 is substituted by glutamic acid (E) (numbering according to        Kabat EU index) and the amino acid at position 213 is        substituted by glutamic acid (E) (numbering according to Kabat        EU index).    -   In an even more particular embodiment, in the constant domain CL        of the first antigen binding moiety the amino acid at position        124 is substituted by lysine (K) (numbering according to Kabat)        and the amino acid at position 123 is substituted by        arginine (R) (numbering according to Kabat), and in the constant        domain CH1 of the first antigen binding moiety the amino acid at        position 147 is substituted by glutamic acid (E) (numbering        according to Kabat EU index) and the amino acid at position 213        is substituted by glutamic acid (E) (numbering according to        Kabat EU index).    -   In particular embodiments, if amino acid substitutions according        to the above embodiments are made in the constant domain CL and        the constant domain CH1 of the first antigen binding moiety, the        constant domain CL of the first antigen binding moiety is of        kappa isotype.    -   Alternatively, the amino acid substitutions according to the        above embodiments may be made in the constant domain CL and the        constant domain CH1 of the second antigen binding moiety instead        of in the constant domain CL and the constant domain CH1 of the        first antigen binding moiety. In particular such embodiments,        the constant domain CL of the second antigen binding moiety is        of kappa isotype.    -   Accordingly, in one embodiment, in the constant domain CL of the        second antigen binding moiety the amino acid at position 124 is        substituted independently by lysine (K), arginine (R) or        histidine (H) (numbering according to Kabat), and in the        constant domain CH1 of the second antigen binding moiety the        amino acid at position 147 or the amino acid at position 213 is        substituted independently by glutamic acid (E), or aspartic        acid (D) (numbering according to Kabat EU index).    -   In a further embodiment, in the constant domain CL of the second        antigen binding moiety the amino acid at position 124 is        substituted independently by lysine (K), arginine (R) or        histidine (H) (numbering according to Kabat), and in the        constant domain CH1 of the second antigen binding moiety the        amino acid at position 147 is substituted independently by        glutamic acid (E), or aspartic acid (D) (numbering according to        Kabat EU index).    -   In still another embodiment, in the constant domain CL of the        second antigen binding moiety the amino acid at position 124 is        substituted independently by lysine (K), arginine (R) or        histidine (H) (numbering according to Kabat) and the amino acid        at position 123 is substituted independently by lysine (K),        arginine (R) or histidine (H) (numbering according to Kabat),        and in the constant domain CH1 of the second antigen binding        moiety the amino acid at position 147 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index) and the amino acid at        position 213 is substituted independently by glutamic acid (E),        or aspartic acid (D) (numbering according to Kabat EU index).    -   In one embodiment, in the constant domain CL of the second        antigen binding moiety the amino acid at position 124 is        substituted by lysine (K) (numbering according to Kabat) and the        amino acid at position 123 is substituted by lysine (K)        (numbering according to Kabat), and in the constant domain CH1        of the second antigen binding moiety the amino acid at position        147 is substituted by glutamic acid (E) (numbering according to        Kabat EU index) and the amino acid at position 213 is        substituted by glutamic acid (E) (numbering according to Kabat        EU index).    -   In another embodiment, in the constant domain CL of the second        antigen binding moiety the amino acid at position 124 is        substituted by lysine (K) (numbering according to Kabat) and the        amino acid at position 123 is substituted by arginine (R)        (numbering according to Kabat), and in the constant domain CH1        of the second antigen binding moiety the amino acid at position        147 is substituted by glutamic acid (E) (numbering according to        Kabat EU index) and the amino acid at position 213 is        substituted by glutamic acid (E) (numbering according to Kabat        EU index).

In a particular embodiment, the bispecific antibody of the inventioncomprises

-   -   I) a first antigen binding moiety that binds to human HLA-G, and        the first antigen binding moiety is a Fab molecule comprising

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ IDNO:32;24

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26;

and

-   -   II) a second antigen binding moiety that binds to human CD3,

wherein the second antigen binding moiety is a Fab molecule wherein thevariable domains VL and VH of the Fab light chain and the Fab heavychain are replaced by each other, comprising

C) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:58 and a VL domain comprising the amino acid sequence of SEQ IDNO:59, or

D) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:66 and a VL domain comprising the amino acid sequence of SEQ IDNO:67, or

E) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:74 and a VL domain comprising the amino acid sequence of SEQ IDNO:75;

and

-   -   III) wherein in the constant domain CL of the first antigen        binding moiety the amino acid at position 124 is substituted        independently by lysine (K), arginine (R) or histidine (H)        (numbering according to Kabat) (in a particular embodiment        independently by lysine (K) or arginine (R)) and the amino acid        at position 123 is substituted independently by lysine (K),        arginine (R) or histidine (H) (numbering according to Kabat) (in        a particular embodiment independently by lysine (K) or arginine        (R)), and in the constant domain CH1 of the first antigen        binding moiety the amino acid at position 147 is substituted        independently by glutamic acid (E), or aspartic acid (D)        (numbering according to Kabat EU index) and the amino acid at        position 213 is substituted independently by glutamic acid (E),        or aspartic acid (D) (numbering according to Kabat EU index).

Bispecific Antibody Formats

The components of the bispecific antibody according to the presentinvention can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIG. 13.

In particular embodiments, the antigen binding moieties comprised in thebispecific antibody are Fab molecules. In such embodiments, the first,second, third etc. antigen binding moiety may be referred to herein asfirst, second, third etc. Fab molecule, respectively.

In one embodiment, the first and the second antigen binding moiety ofthe bispecific antibody are fused to each other, optionally via apeptide linker. In particular embodiments, the first and the secondantigen binding moiety are each a Fab molecule. In one such embodiment,the second antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety. In another such embodiment, the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.In embodiments wherein either (i) the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety or (ii) the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety, additionally the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety maybe fused to each other, optionally via a peptide linker.

A bispecific antibody with a single antigen binding moiety (such as aFab molecule) capable of specific binding to a target cell antigen suchas HLA-G (for example as shown in FIGS. 13A, D, G, H, K, L) is useful,particularly in cases where internalization of the target cell antigenis to be expected following binding of a high affinity antigen bindingmoiety. In such cases, the presence of more than one antigen bindingmoiety specific for the target cell antigen may enhance internalizationof the target cell antigen, thereby reducing its availability.

In other cases, however, it will be advantageous to have a bispecificantibody comprising two or more antigen binding moieties (such as Fabmolecules) specific for a target cell antigen (see examples shown inFIGS. 13B, 13C, 13E, 13F, 13I, 13J, 13M or 13N), for example to optimizetargeting to the target site or to allow crosslinking of target cellantigens.

Accordingly, in particular embodiments, the bispecific antibodyaccording to the present invention comprises a third antigen bindingmoiety.

In one embodiment, the third antigen binding moiety binds to the firstantigen, i.e. HLA-G. In one embodiment, the third antigen binding moietyis a Fab molecule.

In particular embodiments, the third antigen moiety is identical to thefirst antigen binding moiety.

The third antigen binding moiety of the bispecific antibody mayincorporate any of the features, singly or in combination, describedherein in relation to the first antigen binding moiety and/or theantibody that binds HLA-G, unless scientifically clearly unreasonable orimpossible.

In one embodiment, the third antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24; or

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:26; or

In particular embodiments, the third and the first antigen bindingmoiety are each a Fab molecule and the third antigen binding moiety isidentical to the first antigen binding moiety. Thus, in theseembodiments the first and the third antigen binding moiety comprise thesame heavy and light chain amino acid sequences and have the samearrangement of domains (i.e. conventional or crossover)). Furthermore,in these embodiments, the third antigen binding moiety comprises thesame amino acid substitutions, if any, as the first antigen bindingmoiety. For example, the amino acid substitutions described herein as“charge modifications” will be made in the constant domain CL and theconstant domain CH1 of each of the first antigen binding moiety and thethird antigen binding moiety. Alternatively, said amino acidsubstitutions may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety (which in particularembodiments is also a Fab molecule), but not in the constant domain CLand the constant domain CH1 of the first antigen binding moiety and thethird antigen binding moiety.

Like the first antigen binding moiety, the third antigen binding moietyparticularly is a conventional Fab molecule. Embodiments wherein thefirst and the third antigen binding moieties are crossover Fab molecules(and the second antigen binding moiety is a conventional Fab molecule)are, however, also contemplated. Thus, in particular embodiments, thefirst and the third antigen binding moieties are each a conventional Fabmolecule, and the second antigen binding moiety is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CL and CH1 of the Fab heavyand light chains are exchanged/replaced by each other. In otherembodiments, the first and the third antigen binding moieties are each acrossover Fab molecule and the second antigen binding moiety is aconventional Fab molecule.

If a third antigen binding moiety is present, in a particular embodimentthe first and the third antigen moiety bind to human HLA-G, and thesecond antigen binding moiety binds to a second antigen human CD3, mostparticularly CD3 epsilon.

In particular embodiments, the bispecific antibody comprises an Fcdomain composed of a first and a second subunit. The first and thesecond subunit of the Fc domain are capable of stable association.

The bispecific antibody according to the invention can have differentconfigurations, i.e. the first, second (and optionally third) antigenbinding moiety may be fused to each other and to the Fc domain indifferent ways. The components may be fused to each other directly or,preferably, via one or more suitable peptide linkers. Where fusion of aFab molecule is to the N-terminus of a subunit of the Fc domain, it istypically via an immunoglobulin hinge region.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the second antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. In such embodiments, the firstantigen binding moiety may be fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety or to the N-terminus of the other one of the subunits ofthe Fc domain. In particular such embodiments, said first antigenbinding moiety is a conventional Fab molecule, and the second antigenbinding moiety is a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstFab molecule is a crossover Fab molecule and the second Fab molecule isa conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety. In a specificembodiment, the bispecific antibody essentially consists of the firstand the second Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule, andthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the first or the second subunit of the Fcdomain.

Such a configuration is schematically depicted in FIGS. 13G and 13K(with the second antigen binding domain in these examples being a VH/VLcrossover Fab molecule). Optionally, the Fab light chain of the firstFab molecule and the Fab light chain of the second Fab molecule mayadditionally be fused to each other.

In another embodiment, the first and the second antigen binding moietyare each a Fab molecule and the first and the second antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. In a specificembodiment, the bispecific antibody essentially consists of the firstand the second Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thefirst and the second Fab molecule are each fused at the C-terminus ofthe Fab heavy chain to the N-terminus of one of the subunits of the Fcdomain. Such a configuration is schematically depicted in FIGS. 13A and13D (in these examples with the second antigen binding domain being aVH/VL crossover Fab molecule and the first antigen binding moiety beinga conventional Fab molecule). The first and the second Fab molecule maybe fused to the Fc domain directly or through a peptide linker. In aparticular embodiment the first and the second Fab molecule are eachfused to the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region, particularly where the Fc domain is an IgG₁ Fc domain.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain. In such embodiments, the second antigenbinding moiety may be fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second antigen bindingmoiety or (as described above) to the N-terminus of the other one of thesubunits of the Fc domain. In particular such embodiments, said firstantigen binding moiety is a conventional Fab molecule, and the secondantigen binding moiety is a crossover Fab molecule as described herein,i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstFab molecule is a crossover Fab molecule and the second Fab molecule isa conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the second antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding moiety. In a specificembodiment, the bispecific antibody essentially consists of the firstand the second Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thesecond Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first Fab molecule, and thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or the second subunit of the Fc domain. Sucha configuration is schematically depicted in FIGS. 13H and 13L (in theseexamples with the second antigen binding domain being a VH/VL crossoverFab molecule and the first antigen binding moiety being a conventionalFab molecule). Optionally, the Fab light chain of the first Fab moleculeand the Fab light chain of the second Fab molecule may additionally befused to each other.

In some embodiments, a third antigen binding moiety, particularly athird Fab molecule, is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or second subunit of the Fc domain. Inparticular such embodiments, said first and third Fab molecules are eacha conventional Fab molecule, and the second Fab molecule is a crossoverFab molecule as described herein, i.e. a Fab molecule wherein thevariable domains VH and VL or the constant domains CL and CH1 of the Fabheavy and light chains are exchanged/replaced by each other. In othersuch embodiments, said first and third Fab molecules are each acrossover Fab molecule and the second Fab molecule is a conventional Fabmolecule.

In a particular such embodiment, the second and the third antigenbinding moiety are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule. Ina specific embodiment, the bispecific antibody essentially consists ofthe first, the second and the third Fab molecule, the Fc domain composedof a first and a second subunit, and optionally one or more peptidelinkers, wherein the first Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain, and wherein the third Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 13B and 13E (in these examples with the second antigen bindingmoiety being a VH/VL crossover Fab molecule, and the first and the thirdantigen binding moiety being a conventional Fab molecule), and FIGS. 13Jand 13N (in these examples with the second antigen binding moiety beinga conventional Fab molecule, and the first and the third antigen bindingmoiety being a VH/VL crossover Fab molecule). The second and the thirdFab molecule may be fused to the Fc domain directly or through a peptidelinker. In a particular embodiment the second and the third Fab moleculeare each fused to the Fc domain through an immunoglobulin hinge region.In a specific embodiment, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In another such embodiment, the first and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. In a specific embodiment, the bispecific antibody essentiallyconsists of the first, the second and the third Fab molecule, the Fcdomain composed of a first and a second subunit, and optionally one ormore peptide linkers, wherein the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule, and the first Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 13C and 13F (in these examples with the second antigen bindingmoiety being a VH/VL crossover Fab molecule, and the first and the thirdantigen binding moiety being a conventional Fab molecule) and in FIGS.13I and 13M (in these examples with the second antigen binding moietybeing a conventional Fab molecule, and the first and the third antigenbinding moiety being a VH/VL crossover Fab molecule). The first and thethird Fab molecule may be fused to the Fc domain directly or through apeptide linker. In a particular embodiment the first and the third Fabmolecule are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific embodiment, the immunoglobulin hinge region is ahuman IgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In configurations of the bispecific antibody wherein a Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of eachof the subunits of the Fc domain through an immunoglobulin hingeregions, the two Fab molecules, the hinge regions and the Fc domainessentially form an immunoglobulin molecule. In a particular embodimentthe immunoglobulin molecule is an IgG class immunoglobulin. In an evenmore particular embodiment the immunoglobulin is an IgG₁ subclassimmunoglobulin. In another embodiment the immunoglobulin is an IgG₄subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin. In one embodiment, the immunoglobulin comprises a humanconstant region, particularly a human Fc region.

In some of the bispecific antibody of the invention, the Fab light chainof the first Fab molecule and the Fab light chain of the second Fabmolecule are fused to each other, optionally via a peptide linker.Depending on the configuration of the first and the second Fab molecule,the Fab light chain of the first Fab molecule may be fused at itsC-terminus to the N-terminus of the Fab light chain of the second Fabmolecule, or the Fab light chain of the second Fab molecule may be fusedat its C-terminus to the N-terminus of the Fab light chain of the firstFab molecule. Fusion of the Fab light chains of the first and the secondFab molecule further reduces mispairing of unmatched Fab heavy and lightchains, and also reduces the number of plasmids needed for expression ofsome of the bispecific antibodies of the invention.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G₄S)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂. Another suitable such linker comprises the sequence (G₄S)₄.Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where a Fab molecule is fused to theN-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

In certain embodiments the bispecific antibody according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecificantibody further comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In certain embodiments the polypeptides arecovalently linked, e.g., by a disulfide bond.

In certain embodiments the bispecific antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), and a polypeptidewherein the Fab heavy chain of the first Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecific antibodyfurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In certain embodiments the polypeptides arecovalently linked, e.g., by a disulfide bond.

In some embodiments, the bispecific antibody comprises a polypeptidewherein the Fab light chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thefirst Fab molecule, which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). Inother embodiments, the bispecific antibody comprises a polypeptidewherein the Fab heavy chain of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antibody further comprises acrossover Fab light chain polypeptide of the second Fab molecule,wherein the Fab heavy chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the second Fab molecule (VH₍₂₎-CL₍₂₎), and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In others of theseembodiments the bispecific antibody further comprises a polypeptidewherein the Fab heavy chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe first Fab molecule (VH₍₂₎-CL₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptidewherein the Fab light chain polypeptide of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The bispecific antibody according to these embodiments may furthercomprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) apolypeptide wherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain embodiments thepolypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the bispecific antibody comprises a polypeptidewherein the Fab heavy chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain constantregion is replaced by a light chain constant region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thefirst Fab molecule, which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). Inother embodiments, the bispecific antibody comprises a polypeptidewherein the Fab heavy chain of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antibody further comprises acrossover Fab light chain polypeptide of the second Fab molecule,wherein the Fab light chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the second Fab molecule (VL₍₂₎-CH1₍₂₎), and the Fab lightchain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In others ofthese embodiments the bispecific antibody further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe first Fab molecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptidewherein the Fab light chain polypeptide of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The bispecific antibody according to these embodiments may furthercomprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) apolypeptide wherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain embodiments thepolypeptides are covalently linked, e.g., by a disulfide bond.

In all of the different configurations of the bispecific antibodyaccording to the invention, the amino acid substitutions describedherein, if present, may either be in the CH1 and CL domains of the firstand (if present) the third antigen binding moiety/Fab molecule, or inthe CH1 and CL domains of the second antigen binding moiety/Fabmolecule. Preferably, they are in the CH1 and CL domains of the firstand (if present) the third antigen binding moiety/Fab molecule. Inaccordance with the concept of the invention, if amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) antigen binding moiety/Fab molecule, no such aminoacid substitutions are made in the second antigen binding moiety/Fabmolecule. Conversely, if amino acid substitutions as described hereinare made in the second antigen binding moiety/Fab molecule, no suchamino acid substitutions are made in the first (and, if present, thethird) antigen binding moiety/Fab molecule. Amino acid substitutions areparticularly made in bispecific antibodies comprising a Fab moleculewherein the variable domains VL and VH1 of the Fab light chain and theFab heavy chain are replaced by each other.

In particular embodiments of the bispecific antibody according to theinvention, particularly wherein amino acid substitutions as describedherein are made in the first (and, if present, the third) antigenbinding moiety/Fab molecule, the constant domain CL of the first (and,if present, the third) Fab molecule is of kappa isotype. In otherembodiments of the bispecific antibody according to the invention,particularly wherein amino acid substitutions as described herein aremade in the second antigen binding moiety/Fab molecule, the constantdomain CL of the second antigen binding moiety/Fab molecule is of kappaisotype. In some embodiments, the constant domain CL of the first (and,if present, the third) antigen binding moiety/Fab molecule and theconstant domain CL of the second antigen binding moiety/Fab molecule areof kappa isotype.

In a particular aspect, the invention provides a bispecific antibodycomprising

a) a first and a third antigen binding moiety that binds to a firstantigen; wherein the first antigen is HLA-G, and wherein the first andthe second antigen binding moiety are each a (conventional) Fab moleculecomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 7 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 24,

b) a second antigen binding moiety that binds to a second antigen;wherein the second antigen is CD3 and wherein the second antigen bindingmoiety is Fab molecule wherein the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,comprising (i) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 58 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 59; or (ii) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 66 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:67; or (iii) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 74 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 75; and

c) an Fc domain composed of a first and a second subunit;

wherein

-   -   in the constant domain CL of the first and the third antigen        binding moiety under a) the amino acid at position 124 is        substituted by lysine (K) (numbering according to Kabat) and the        amino acid at position 123 is substituted by lysine (K) or        arginine (R) (numbering according to Kabat) (most particularly        by arginine (R)), and wherein in the constant domain CH1 of the        first and the third antigen binding moiety under a) the amino        acid at position 147 is substituted by glutamic acid (E)        (numbering according to Kabat EU index) and the amino acid at        position 213 is substituted by glutamic acid (E) (numbering        according to Kabat EU index);

and wherein further

-   -   the first antigen binding moiety under a) is fused at the        C-terminus of the Fab heavy chain to the N-terminus of the Fab        heavy chain of the second antigen binding moiety under b), and        the second antigen binding moiety under b) and the third antigen        binding moiety under a) are each fused at the C-terminus of the        Fab heavy chain to the N-terminus of one of the subunits of the        Fc domain under c).

In one embodiment according to these aspects of the invention, in thefirst subunit of the Fc domain the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the second subunit ofthe Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V) and optionally the threonine residue at position366 is replaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) (numberingsaccording to Kabat EU index).

In a further embodiment according to these aspects of the invention, inthe first subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue (E356C)(particularly the serine residue at position 354 is replaced with acysteine residue), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).

In still a further embodiment according to these aspects of theinvention, in each of the first and the second subunit of the Fc domainthe leucine residue at position 234 is replaced with an alanine residue(L234A), the leucine residue at position 235 is replaced with an alanineresidue (L235A) and the proline residue at position 329 is replaced by aglycine residue (P329G) (numbering according to Kabat EU index).

In still a further embodiment according to these aspects of theinvention, the Fc domain is a human IgG₁ Fc domain.

A specific embodiment of the invention is a bispecific antibody thatbinds to human HLA-G and to human CD3 wherein the antibody comprises apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 76, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 77, a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 78, and a polypeptide comprising an amino acid sequence that isat least 98%, or 99% identical to the sequence of SEQ ID NO: 79 (whereinin the VH or VL framework regions or in the constant regions amino acidsare substituted without affecting the specific binding properties andthe properties of constant regions of such bispecific antibody)

A specific embodiment of the invention is a bispecific antibody thatbinds to human HLA-G and to human CD3 wherein the antibody comprises apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 76, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 77, a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 78, and a polypeptide comprising an amino acid sequence that isat least 98%, or 99% identical to the sequence of SEQ ID NO: 79,

and wherein the bispecific antibody has one or more of the of thefollowing properties:

the bispecific antibody shows

a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see Example 10);and/or

b) antibody mediated IFN gamma secretion by T cells on SKOV3 cellstransfected with recombinant HLA-G (SKOV3 HLA-G) and/or on JEG3 cellsexpressing endogenous HLA-G wherein the IFN gamma secretion was detected(by Luminex technology) (see Example 11); and or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cellstransfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3 cellsexpressing endogenous HLA-G wherein the cytotoxicity was detected bymeasuring Caspase 8 activation in cells after treatment with bispecificantibody (see Example 12); and/or

-   -   d) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example        13); and/or

e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (seeExample 14).

In a further specific embodiment, the bispecific antibody comprises apolypeptide comprising the amino acid sequence of SEQ ID NO: 76, apolypeptide comprising the amino acid sequence of SEQ ID NO: 77, apolypeptide comprising the amino acid sequence of SEQ ID NO: 78 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 79.

A further specific embodiment of the invention is a bispecific antibodythat binds to human HLA-G and to human CD3 wherein the antibodycomprises a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 80, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 81, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 82, and a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 83 (wherein in the VH or VL framework regions or in the constantregions amino acids are substituted without affecting the specificbinding properties and the properties of constant regions of suchbispecific antibody)

A specific embodiment of the invention is a bispecific antibody thatbinds to human HLA-G and to human CD3 wherein the antibody comprises apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 80, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 81, a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 82, and a polypeptide comprising an amino acid sequence that isat least 98%, or 99% identical to the sequence of SEQ ID NO: 83,

and wherein the bispecific antibody has one or more of the of thefollowing properties:

the bispecific antibody shows

a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see Example 10);and/or

b) antibody mediated IFN gamma secretion by T cells on SKOV3 cellstransfected with recombinant HLA-G (SKOV3 HLA-G) and/or on JEG3 cellsexpressing endogenous HLA-G wherein the IFN gamma secretion was detected(by Luminex technology) (see Example 11); and or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cellstransfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3 cellsexpressing endogenous HLA-G wherein the cytotoxicity was detected bymeasuring Caspase 8 activation in cells after treatment with bispecificantibody (see Example 12); and/or

-   -   d) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example        13); and/or

e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (seeExample 14).

In a further specific embodiment, the bispecific antibody comprises apolypeptide comprising the amino acid sequence of SEQ ID NO: 80, apolypeptide comprising the amino acid sequence of SEQ ID NO: 81, apolypeptide comprising the amino acid sequence of SEQ ID NO: 82 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 83.

A further specific embodiment of the invention is a bispecific antibodythat binds to human HLA-G and to human CD3 wherein the antibodycomprises a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 84, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 85, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 86, and a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 87 (wherein in the VH or VL framework regions or in the constantregions amino acids are substituted without affecting the specificbinding properties and the properties of constant regions of suchbispecific antibody)

A specific embodiment of the invention is a bispecific antibody thatbinds to human HLA-G and to human CD3 wherein the antibody comprises apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 84, a polypeptide comprisingan amino acid sequence that is at least 98%, or 99% identical to thesequence of SEQ ID NO: 85, a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 86, and a polypeptide comprising an amino acid sequence that isat least 98%, or 99% identical to the sequence of SEQ ID NO: 87,

and wherein the bispecific antibody has one or more of the of thefollowing properties:

the bispecific antibody shows

a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see Example 10);and/or

b) antibody mediated IFN gamma secretion by T cells on SKOV3 cellstransfected with recombinant HLA-G (SKOV3 HLA-G) and/or on JEG3 cellsexpressing endogenous HLA-G wherein the IFN gamma secretion was detected(by Luminex technology) (see Example 11); and or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cellstransfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3 cellsexpressing endogenous HLA-G wherein the cytotoxicity was detected bymeasuring Caspase 8 activation in cells after treatment with bispecificantibody (see Example 12); and/or

-   -   d) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example        13); and/or

e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (seeExample 14).

In a further specific embodiment, the bispecific antibody comprises apolypeptide comprising the amino acid sequence of SEQ ID NO: 84, apolypeptide comprising the amino acid sequence of SEQ ID NO: 85, apolypeptide comprising the amino acid sequence of SEQ ID NO: 86 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 87.

Fc Domain

In particular embodiments, the bispecific antibody of the inventioncomprises an Fc domain composed of a first and a second subunit. It isunderstood, that the features of the Fc domain described herein inrelation to the bispecific antibody can equally apply to an Fc domaincomprised in a monospecific anti-HLAG antibody of the invention exceptfor those modifications relevant for Fc heterodimerization.

The Fc domain of the bispecific antibody consists of a pair ofpolypeptide chains comprising heavy chain domains of an immunoglobulinmolecule. For example, the Fc domain of an immunoglobulin G (IgG)molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgGheavy chain constant domains. The two subunits of the Fc domain arecapable of stable association with each other. In one embodiment, thebispecific antibody of the invention comprises not more than one Fcdomain.

In one embodiment, the Fc domain of the bispecific antibody is an IgG Fcdomain. In a particular embodiment, the Fc domain is an IgG₁ Fc domain.In another embodiment the Fc domain is an IgG₄ Fc domain. In a morespecific embodiment, the Fc domain is an IgG₄ Fc domain comprising anamino acid substitution at position S228 (Kabat EU index numbering),particularly the amino acid substitution S228P. This amino acidsubstitution reduces in vivo Fab arm exchange of IgG₄ antibodies (seeStubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).In a further particular embodiment, the Fc domain is a human Fc domain.In an even more particular embodiment, the Fc domain is a human IgG₁ Fcdomain.

Fc Domain Modifications Promoting Heterodimerization

Bispecific antibodies according to the invention comprise differentantigen binding moieties, which may be fused to one or the other of thetwo subunits of the Fc domain, thus the two subunits of the Fc domainare typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of bispecific antibodiesin recombinant production, it will thus be advantageous to introduce inthe Fc domain of the bispecific antibody a modification promoting theassociation of the desired polypeptides.

Accordingly, in particular embodiments, the Fc domain of the bispecificantibody according to the invention comprises a modification promotingthe association of the first and the second subunit of the Fc domain.The site of most extensive protein-protein interaction between the twosubunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one embodiment said modification is in the CH3 domain of the Fcdomain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomdimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (e.g. VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the bispecific antibody which reduce heavy/light chainmispairing and Bence Jones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the bispecific antibody an amino acidresidue is replaced with an amino acid residue having a larger sidechain volume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a smaller side chain volume, thereby generating acavity within the CH3 domain of the second subunit within which theprotuberance within the CH3 domain of the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in (the CH3 domain of) the first subunit ofthe Fc domain (the “knobs” subunit) the threonine residue at position366 is replaced with a tryptophan residue (T366W), and in (the CH3domain of) the second subunit of the Fc domain (the “hole” subunit) thetyrosine residue at position 407 is replaced with a valine residue(Y407V). In one embodiment, in the second subunit of the Fc domainadditionally the threonine residue at position 366 is replaced with aserine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A) (numberings according to KabatEU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C) (particularly the serineresidue at position 354 is replaced with a cysteine residue), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingsaccording to Kabat EU index). Introduction of these two cysteineresidues results in formation of a disulfide bridge between the twosubunits of the Fc domain, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesthe amino acid substitutions S354C and T366W, and the second subunit ofthe Fc domain comprises the amino acid substitutions Y349C, T366S, L368Aand Y407V (numbering according to Kabat EU index).

In a particular embodiment the antigen binding moiety that binds to thesecond antigen (e.g. an activating T cell antigen) is fused (optionallyvia the first antigen binding moiety, which binds to HLA-G, and/or apeptide linker) to the first subunit of the Fc domain (comprising the“knob” modification). Without wishing to be bound by theory, fusion ofthe antigen binding moiety that binds a second antigen, such as anactivating T cell antigen, to the knob-containing subunit of the Fcdomain will (further) minimize the generation of antibodies comprisingtwo antigen binding moieties that bind to an activating T cell antigen(steric clash of two knob-containing polypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment, the heterodimerization approach described in EP1870459, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the bispecificantibody of the invention are amino acid mutations R409D; K370E in oneof the two CH3 domains (of the Fc domain) and amino acid mutationsD399K; E357K in the other one of the CH3 domains of the Fc domain(numbering according to Kabat EU index).

In another embodiment, the bispecific antibody of the inventioncomprises amino acid mutation T366W in the CH3 domain of the firstsubunit of the Fc domain and amino acid mutations T366S, L368A, Y407V inthe CH3 domain of the second subunit of the Fc domain, and additionallyamino acid mutations R409D; K370E in the CH3 domain of the first subunitof the Fc domain and amino acid mutations D399K; E357K in the CH3 domainof the second subunit of the Fc domain (numberings according to Kabat EUindex).

In another embodiment, the bispecific antibody of the inventioncomprises amino acid mutations S354C, T366W in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations Y349C, T366S,L368A, Y407V in the CH3 domain of the second subunit of the Fc domain,or said bispecific antibody comprises amino acid mutations Y349C, T366Win the CH3 domain of the first subunit of the Fc domain and amino acidmutations S354C, T366S, L368A, Y407V in the CH3 domains of the secondsubunit of the Fc domain and additionally amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain and aminoacid mutations D399K; E357K in the CH3 domain of the second subunit ofthe Fc domain (all numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment, a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment, the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment, the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment, a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment, the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment, a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the bispecific antibody or its Fc domain is of IgG₂subclass and the heterodimerization approach described in WO 2010/129304is used alternatively.

In an alternative embodiment, a modification promoting association ofthe first and the second subunit of the Fc domain comprises amodification mediating electrostatic steering effects, e.g. as describedin PCT publication WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment, a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment, the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment, the heterodimerization approach describedin WO 2007/147901 is used alternatively. In one embodiment, a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach describedin WO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the bispecific antibody (or the antibody)favorable pharmacokinetic properties, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibody (orthe antibody) to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Moreover, the co-activation of Fcreceptor signaling pathways may lead to cytokine secretion/releasewhich, in combination with the T cell activating properties (e.g. inembodiments of the bispecific antibody wherein the second antigenbinding moiety binds to an activating T cell antigen) and the longhalf-life of the bispecific antibody, results in excessive activation ofcytokine receptors and severe side effects upon systemic administration.Activation of (Fc receptor-bearing) immune cells other than T cells mayeven reduce efficacy of the bispecific antibody (particularly abispecific antibody wherein the second antigen binding moiety binds toan activating T cell antigen) due to the potential destruction of Tcells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the bispecificantibody according to the invention exhibits reduced binding affinity toan Fc receptor and/or reduced effector function, as compared to a nativeIgG₁ Fc domain. In one such embodiment the Fc domain (or the bispecificantibody comprising said Fc domain) exhibits less than 50%, preferablyless than 20%, more preferably less than 10% and most preferably lessthan 5% of the binding affinity to an Fc receptor, as compared to anative IgG₁ Fc domain (or a bispecific antibody comprising a native IgG₁Fc domain), and/or less than 50%, preferably less than 20%, morepreferably less than 10% and most preferably less than 5% of theeffector function, as compared to a native IgG₁ Fc domain domain (or abispecific antibody comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the bispecific antibody comprisingsaid Fc domain) does not substantially bind to an Fc receptor and/orinduce effector function. In a particular embodiment the Fc receptor isan Fcγ receptor. In one embodiment the Fc receptor is a human Fcreceptor. In one embodiment the Fc receptor is an activating Fcreceptor. In a specific embodiment the Fc receptor is an activatinghuman Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. In one embodiment the effectorfunction is one or more selected from the group of CDC, ADCC, ADCP, andcytokine secretion. In a particular embodiment, the effector function isADCC. In one embodiment, the Fc domain domain exhibits substantiallysimilar binding affinity to neonatal Fc receptor (FcRn), as compared toa native IgG₁ Fc domain domain. Substantially similar binding to FcRn isachieved when the Fc domain (or the bispecific antibody comprising saidFc domain) exhibits greater than about 70%, particularly greater thanabout 80%, more particularly greater than about 90% of the bindingaffinity of a native IgG₁ Fc domain (or the bispecific antibodycomprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the bispecific antibody comprises one or more amino acidmutation that reduces the binding affinity of the Fc domain to an Fcreceptor and/or effector function. Typically, the same one or more aminoacid mutation is present in each of the two subunits of the Fc domain.In one embodiment, the amino acid mutation reduces the binding affinityof the Fc domain to an Fc receptor. In one embodiment, the amino acidmutation reduces the binding affinity of the Fc domain to an Fc receptorby at least 2-fold, at least 5-fold, or at least 10-fold. In embodimentswhere there is more than one amino acid mutation that reduces thebinding affinity of the Fc domain to the Fc receptor, the combination ofthese amino acid mutations may reduce the binding affinity of the Fcdomain to an Fc receptor by at least 10-fold, at least 20-fold, or evenat least 50-fold. In one embodiment the bispecific antibody comprisingan engineered Fc domain exhibits less than 20%, particularly less than10%, more particularly less than 5% of the binding affinity to an Fcreceptor as compared to a bispecific antibody comprising anon-engineered Fc domain. In a particular embodiment, the Fc receptor isan Fcγ receptor. In some embodiments, the Fc receptor is a human Fcreceptor. In some embodiments, the Fc receptor is an activating Fcreceptor. In a specific embodiment, the Fc receptor is an activatinghuman Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. Preferably, binding to each of thesereceptors is reduced. In some embodiments, binding affinity to acomplement component, specifically binding affinity to C1q, is alsoreduced. In one embodiment, binding affinity to neonatal Fc receptor(FcRn) is not reduced. Substantially similar binding to FcRn, i.e.preservation of the binding affinity of the Fc domain to said receptor,is achieved when the Fc domain (or the bispecific antibody comprisingsaid Fc domain) exhibits greater than about 70% of the binding affinityof a non-engineered form of the Fc domain (or the bispecific antibodycomprising said non-engineered form of the Fc domain) to FcRn. The Fcdomain, or bispecific antibodies of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments, the Fc domain of thebispecific antibody is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming In one embodiment, the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment, the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a bispecific antibody comprising anon-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment, the Fc domain comprisesan amino acid substitution at a position selected from the group ofE233, L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment, the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodiments,the Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment, the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment, the amino acidsubstitution is P329A or P329G, particularly P329G (numberings accordingto Kabat EU index). In one embodiment, the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionat a position selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment, the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments, the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments, the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA”or “LALAPG”). Specifically, in particular embodiments, each subunit ofthe Fc domain comprises the amino acid substitutions L234A, L235A andP329G (Kabat EU index numbering), i.e. in each of the first and thesecond subunit of the Fc domain the leucine residue at position 234 isreplaced with an alanine residue (L234A), the leucine residue atposition 235 is replaced with an alanine residue (L235A) and the prolineresidue at position 329 is replaced by a glycine residue (P329G)(numbering according to Kabat EU index).

In one such embodiment, the Fc domain is an IgG₁ Fc domain, particularlya human IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, which is incorporated herein byreference in its entirety. WO 2012/130831 also describes methods ofpreparing such mutant Fc domains and methods for determining itsproperties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments, the Fc domain of the bispecific antibodies of theinvention is an IgG₄ Fc domain, particularly a human IgG₄ Fc domain. Inone embodiment, the IgG₄ Fc domain comprises amino acid substitutions atposition S228, specifically the amino acid substitution S228P(numberings according to Kabat EU index). To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment, the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E(numberings according to Kabat EU index). In another embodiment, theIgG₄ Fc domain comprises an amino acid substitution at position P329,specifically the amino acid substitution P329G (numberings according toKabat EU index). In a particular embodiment, the IgG₄ Fc domaincomprises amino acid substitutions at positions S228, L235 and P329,specifically amino acid substitutions S228P, L235E and P329G (numberingsaccording to Kabat EU index). Such IgG₄ Fc domain mutants and their Fcγreceptor binding properties are described in PCT publication no. WO2012/130831, incorporated herein by reference in its entirety.

In a particular embodiment, the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or bispecific antibodies comprising an Fc domain for Fcreceptors may be evaluated using cell lines known to express particularFc receptors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a bispecific antibody comprisingan Fc domain, can be measured by methods known in the art. Examples ofin vitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the Fc domain, or the bispecificantibody comprising the Fc domain, is able to bind C1q and hence has CDCactivity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance/half life determinations can also beperformed using methods known in the art (see, e.g., Petkova, S. B. etal., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929).

In a further aspect, an anti-HLA-G antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-6 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant KD of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one preferred embodiment, KD is measured using surface plasmonresonance assays using a BIACORE®) at 25° C. with immobilized antigenCMS chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on) or ka) and dissociation rates(k_(off) or kd) are calculated using a simple one-to-one Langmuirbinding model (BIACORE ® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant KD is calculated as the ratio kd/ka(k_(off)/k_(on.)) See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881. If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson, P. J. etal., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see,e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies,Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994),pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134; andHolliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J. et al.,Nat. Med. 9 (20039 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parental antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which CDRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the CDR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5, 821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingSDR (a-CDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498(describing “resurfacing”); Dall'Acqua, W. F. et al., Methods 36 (2005)43-60 (describing “FR shuffling”); and Osbourn, J. et al., Methods 36(2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, M. A. and vande Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147(1991) 86-95) Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37and further described, e.g., in the McCafferty, J. et al., Nature 348(1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks,J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. andBradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol.Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284(2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G. et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths,A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and USPatent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the CDRs and FRs (in a preferred embodimentframework residues not relevant for the binding properties of theantibodies (see e.g. (see e.g. Foote J. and Winter G., J. Mol. Biol.(1992) 224, 487-499). Exemplary changes are provided in Table 1 underthe heading of “exemplary substitutions”, and as further described belowin reference to amino acid side chain classes. Conservativesubstitutions are shown in Table 1 under the heading of “preferredsubstitutions”. Amino acid substitutions may be introduced into anantibody of interest and the products screened for a desired activity,e.g., retained/improved antigen binding, decreased immunogenicity, orimproved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or moreCDRs of a parent antibody (e.g. a humanized or human antibody).Generally, the resulting variant(s) selected for further study will havemodifications (e.g., improvements) in certain biological properties(e.g., increased affinity, reduced immunogenicity) relative to theparent antibody and/or will have substantially retained certainbiological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore CDR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improveantibody affinity. Such alterations may be made in CDR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), with theresulting variant VH or VL being tested for binding affinity. Affinitymaturation by constructing and reselecting from secondary libraries hasbeen described, e.g., in Hoogenboom, H. R. et al. in Methods inMolecular Biology 178 (2002) 1-37. In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves CDR-directed approaches, in which several CDRresidues (e.g., 4-6 residues at a time) are randomized CDR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more CDRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in CDRs. Such alterations may be outside of CDR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each CDR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604)

In one embodiment the invention such antibody is a IgG1 with mutationsL234A and L235A or with mutations L234A, L235A and P329G. In anotherembodiment or IgG4 with mutations S228P and L235E or S228P, L235E or andP329G (numbering according to EU index of Kabat et al, Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991)

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

c) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

d) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-HLA-G antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one preferred embodiment,the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, aHEK293 cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-HLA-G antibody or bispecificantibody is provided, wherein the method comprises culturing a host cellcomprising a nucleic acid encoding the antibody, as provided above,under conditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-HLA-G antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

The most suitable host cells for cloning or expression ofantibody-encoding vectors eukaryotic cells, preferably mammalian cells,described herein.

Vertebrate cells may be used as hosts. For example, mammalian cell linesthat are adapted to grow in suspension may be useful. Other examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7); human embryonic kidney line (293 or 293 cells asdescribed, e.g., in Graham, F. L. et al., J. Gen Virol. 36 (1977)59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cellsas described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980) 243-252);monkey kidney cells (CV1); African green monkey kidney cells (VERO-76);human cervical carcinoma cells (HELA); canine kidney cells (MDCK;buffalo rat liver cells (BRL 3A); human lung cells (W138); human livercells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Most useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

To some extent also prokaryotic cells may be used, however with thedisadavantage of sometimes higher efforts and more complex procedures.For expression of antibody fragments and polypeptides in bacteria, see,e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See alsoCharlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B. K.C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254, describingexpression of antibody fragments in E. coli.) After expression, theantibody may be isolated from the bacterial cell paste in a solublefraction and can be further purified.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

C. Assays

Anti-HLA-G antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris, G. E. (ed.), Epitope MappingProtocols, In: Methods in Molecular Biology, Vol. 66, Humana Press,Totowa, N.J. (1996).

2. Activity Assays

In one aspect, assays are provided for identifying anti-HLA-G antibodiesthereof having biological activity. Biological activity may include,e.g., the ability to enhance the activation and/or proliferation ofdifferent immune cells including T-cells. E.g. they enhance secretion ofimmunomodulating cytokines (e.g. interferon-gamma (IFN-gamma) and/ortumor necrosis factor alpha (TNF alpha)). Other immunomodulatingcytokines which are or can be enhance are e.g IL1B, IL6, IL12, GranzymeB etc. binding to different cell types. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity as described e.g. in Examples below.

D. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HLA-G antibodies provided hereinis useful for detecting the presence of HLA-G in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as immune cell or T cell infiltratesand or tumor cells.

In one embodiment, an anti-HLA-G antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HLA-G in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-HLA-G antibody as described herein under conditionspermissive for binding of the anti-HLA-G antibody to HLA-G, anddetecting whether a complex is formed between the anti-HLA-G antibodyand HLA-G. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-HLA-G antibody is used to select subjects eligiblefor therapy with an anti-HLA-G antibody, e.g. where HLA-G is a biomarkerfor selection of patients.

In certain embodiments, labeled anti-HLA-G antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

E. Pharmaceutical Formulations

Pharmaceutical formulations of anti-HLA-G antibodies or theanti-HLA-G/anti-CD3 bispecific antibodies as described herein areprepared by mixing such antibodies having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.)(1980)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

F. Therapeutic Methods and Compositions

Any of the anti-HLA-G antibodies or the anti-HLA-G/anti-CD3 bispecificantibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-HLA-G antibody or an anti-HLA-G/anti-CD3bispecific antibody for use as a medicament is provided. In furtheraspects, an anti-HLA-G antibody or an anti-HLA-G/anti-CD3 bispecificantibody for use in treating cancer is provided. In certain embodiments,an anti-HLA-G antibody or an anti-HLA-G/anti-CD3 bispecific antibody foruse in a method of treatment is provided. In certain embodiments, theinvention provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD3bispecific antibody for use in a method of treating an individual havingcancer comprising administering to the individual an effective amount ofthe anti-HLA-G/anti-CD3 bispecific antibody.

In further embodiments, the invention provides an anti-HLA-G antibody oran anti-HLA-G/anti-CD3 bispecific antibody for use as immunomodulatoryagent/to directly or indirectly induce proliferation and/or activationof immune cells (like T cells, B cells and myeloid cells includingmonocytes, macrophages, dendritic cells, plasmacytoid dendritic cells)e.g. by secretion of immunostimulatory cytokines like TNFalpha (TNFa)and IFNgamma (IFNg) or further recruitment of immune cells. In certainembodiments, the invention provides an anti-HLA-G antibody or ananti-HLA-G/anti-CD3 bispecific antibody for use in a method ofimmunomodulatory agent/to directly or indirectly induce proliferation,activation of immune cells e.g. by secretion of immunostimulatorycytokines like TNFa and IFNgamma or further recruitment of immune cellsin an individual comprising administering to the individual an effectiveof the anti-HLA-G antibody or anti-HLA-G/anti-CD3 bispecific antibodyfor immunomodulation/or directly or indirectly induce proliferation,activation of immune cells e.g. by secretion of immunostimulatorycytokines like TNFa and IFNgamma or further recruitment of immune cells.

In further embodiments, the invention provides an anti-HLA-G antibody oran anti-HLA-G/anti-CD3 bispecific antibody for use as immunostimmulatoryagent/or stimulating tumor necrosis factor alpha (TNF alpha) secretion.In certain embodiments, the invention provides an anti-HLA-G antibody oran anti-HLA-G/anti-CD3 bispecific antibody for use in a method ofimmunomodulation to directly or indirectly induce proliferation,activation e.g. by secretion of immunostimulatory cytokines like TNFaand IFNg or further recruitment of immune cells in an individualcomprising administering to the individual an effective of theanti-HLA-G antibody or anti-HLA-G/anti-CD3 bispecific antibodyimmunomodulation to directly or indirectly induce proliferation,activation e.g. by secretion of immunostimulatory cytokines like TNFaand IFNg or further recruitment of immune cells

The term “cancer” as used herein may be, for example, lung cancer, nonsmall cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, neoplasms of the central nervous system (CNS), spinal axistumors, brain stem glioma, glioblastoma multiforme, astrocytomas,schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cellcarcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, includingrefractory versions of any of the above cancers, or a combination of oneor more of the above cancers.

An “individual” according to any of the above embodiments is preferablya human. In a further aspect, the invention provides for the use of ananti-HLA-G antibody in the manufacture or preparation of a medicament.In one embodiment, the medicament is for treatment of cancer. In afurther embodiment, the medicament is for use in a method of treatingcancer comprising administering to an individual having cancer aneffective amount of the medicament. In a further embodiment, themedicament is for inducing cell mediated lysis of cancer cells In afurther embodiment, the medicament is for use in a method of inducingcell mediated lysis of cancer cells in an individual suffering fromcancer comprising administering to the individual an amount effective ofthe medicament to induce apoptosis in a cancer cell/or to inhibit cancercell proliferation. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having cancer an effective amount of an anti-HLA-G antibodyor an anti-HLA-G/anti-CD3 bispecific antibody. An “individual” accordingto any of the above embodiments may be a human.

In a further aspect, the invention provides a method for inducing cellmediated lysis of cancer cells in an individual suffering from cancer.In one embodiment, the method comprises administering to the individualan effective amount of an anti-HLA-G antibody or an anti-HLA-G/anti-CD3bispecific antibody to induce cell mediated lysis of cancer cells in theindividual suffering from cancer. In one embodiment, an “individual” isa human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-HLA-G antibodies or anti-HLA-G/anti-CD3bispecific antibodies provided herein, e.g., for use in any of the abovetherapeutic methods. In one embodiment, a pharmaceutical formulationcomprises any of the anti-HLA-G antibodies or anti-HLA-G/anti-CD3bispecific antibodies provided herein and a pharmaceutically acceptablecarrier.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intra-arterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-HLA-G antibody or ananti-HLA-G/anti-CD3 bispecific antibody.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-HLA-G antibody or ananti-HLA-G/anti-CD3 bispecific antibody.

II. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Description of the Amino Acid Sequences

Anti-HLA-G Antibodies/Antigen Binding Moieties (SEQ ID Nos of VariableRegions and Complement Determining Regions (CDRs)):

-   -   SEQ ID NO: 1 heavy chain CDR-H1, HLA-G-0090    -   SEQ ID NO: 2 heavy chain CDR-H2, HLA-G-0090    -   SEQ ID NO: 3 heavy chain CDR-H3, HLA-G-0090    -   SEQ ID NO: 4 light chain CDR-L1, HLA-G-0090    -   SEQ ID NO: 5 light chain CDR-L2, HLA-G-0090    -   SEQ ID NO: 6 light chain CDR-L3, HLA-G-0090    -   SEQ ID NO: 7 heavy chain variable domain VH, HLA-G-0090    -   SEQ ID NO: 8 light chain variable domain VL, HLA-G-0090    -   SEQ ID NO: 9 light chain CDR-L1, HLA-G-0090-VL-N31D    -   SEQ ID NO: 10 light chain variable domain VL, HLA-G-0090-VL-N31D    -   SEQ ID NO: 11 light chain CDR-L1, HLA-G-0090-VL-N31L    -   SEQ ID NO: 12 light chain variable domain VL, HLA-G-0090-VL-N31L    -   SEQ ID NO: 13 light chain CDR-L1, HLA-G-0090-VL-N31Q    -   SEQ ID NO: 14 light chain variable domain VL, HLA-G-0090-VL-N31Q    -   SEQ ID NO: 15 light chain CDR-L1, HLA-G-0090-VL-N31S    -   SEQ ID NO: 16 light chain variable domain VL, HLA-G-0090-VL-N31S    -   SEQ ID NO: 17 light chain CDR-L1, HLA-G-0090-VL-N31T    -   SEQ ID NO: 18 light chain variable domain VL, HLA-G-0090-VL-N31T    -   SEQ ID NO: 19 light chain CDR-L1, HLA-G-0090-VL-N31Y    -   SEQ ID NO: 20 light chain variable domain VL, HLA-G-0090-VL-N31Y    -   SEQ ID NO: 21 light chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y    -   SEQ ID NO: 22 light chain variable domain VL,        HLA-G-0090-VL-N31Y-N38Y    -   SEQ ID NO: 23 light chain CDR-L1, HLA-G-0090-VL-S32P    -   SEQ ID NO: 24 light chain variable domain VL, HLA-G-0090-VL-S32P    -   SEQ ID NO: 25 light chain CDR-L1, HLA-G-0090-VL-S33A    -   SEQ ID NO: 26 light chain variable domain VL, HLA-G-0090-VL-S33A    -   SEQ ID NO: 27 light chain CDR-L1, HLA-G-0090-VL-S33D    -   SEQ ID NO: 28 light chain variable domain VL, HLA-G-0090-VL-S33D    -   SEQ ID NO: 29 light chain CDR-L1, HLA-G-0090-VL-S33P    -   SEQ ID NO: 30 light chain variable domain VL, HLA-G-0090-VL-S33P

Further Sequences

-   -   SEQ ID NO: 31 exemplary human HLA-G    -   SEQ ID NO: 32 exemplary human HLA-G extracellular domain (ECD)    -   SEQ ID NO: 33 exemplary human β2M    -   SEQ ID NO: 34 modified human HLA-G (wherein the HLA-G specific        amino acids have been replaced by HLA-A consensus amino acids        (=degrafted HLA-G see also FIG. 1) ECD)    -   SEQ ID NO: 35 exemplary human HLA-A2    -   SEQ ID NO: 36 exemplary human HLA-A2 ECD    -   SEQ ID NO: 37 exemplary mouse H2Kd ECD    -   SEQ ID NO: 38 exemplary rat RT1A ECD    -   SEQ ID NO: 39 exemplary human HLA-G β2M MHC class I complex    -   SEQ ID NO: 40 exemplary modified human HLA-G β2M MHC class I        complex (wherein the HLA-G specific amino acids have been        replaced by HLA-A consensus amino acids (=degrafted HLA-G) see        also FIG. 2)    -   SEQ ID NO: 41 exemplary mouse H2Kd β2M MHC class I complex    -   SEQ ID NO: 42 exemplary human HLA-G/mouse H2Kd β2M MHC class I        complex wherein the positions specific for human HLA-G are        grafted onto the mouse H2Kd framework    -   SEQ ID NO: 43 exemplary rat RT1A β2M MHC class I complex    -   SEQ ID NO: 44 exemplary human HLA-G/rat RT1A β2M MHC class I        complex wherein the positions specific for human HLA-G are        grafted onto the rat RT1A framework    -   SEQ ID NO: 45 linker and his-Tag    -   SEQ ID NO: 46 peptide    -   SEQ ID NO: 47 human kappa light chain constant region    -   SEQ ID NO: 48 human lambda light chain constant region    -   SEQ ID NO: 49 human heavy chain constant region derived from        IgG1    -   SEQ ID NO: 50 human heavy chain constant region derived from        IgG1 with mutations L234A, L235A and P329G    -   SEQ ID NO: 51 human heavy chain constant region derived from        IgG4

Anti-CD3 Antibodies/Antigen Binding Moieties (Variable Regions andComplementarity Determining Regions (CDRs)):

-   -   SEQ ID NO: 52 heavy chain CDR-H1, P035-093 (abbreviated as P035)    -   SEQ ID NO: 53 heavy chain CDR-H2, P035-093    -   SEQ ID NO: 54 heavy chain CDR-H3, P035-093    -   SEQ ID NO: 55 light chain CDR-L1, P035-093    -   SEQ ID NO: 56 light chain CDR-L2, P035-093    -   SEQ ID NO: 57 light chain CDR-L3, P035-093    -   SEQ ID NO: 58 heavy chain variable domain VH, P035-093    -   SEQ ID NO: 59 light chain variable domain VL, P035-093    -   SEQ ID NO: 60 heavy chain CDR-H1, Clone 22 (abbreviated as C122)    -   SEQ ID NO: 61 heavy chain CDR-H2, Clone 22    -   SEQ ID NO: 62 heavy chain CDR-H3, Clone 22    -   SEQ ID NO: 63 light chain CDR-L1, Clone 22    -   SEQ ID NO: 64 light chain CDR-L2, Clone 22    -   SEQ ID NO: 65 light chain CDR-L3, Clone 22    -   SEQ ID NO: 66 heavy chain variable domain VH, Clone 22    -   SEQ ID NO: 67 light chain variable domain VL, Clone 22    -   SEQ ID NO: 68 heavy chain CDR-H1, V9    -   SEQ ID NO: 69 heavy chain CDR-H2, V9    -   SEQ ID NO: 70 heavy chain CDR-H3, V9    -   SEQ ID NO: 71 light chain CDR-L1, V9    -   SEQ ID NO: 72 light chain CDR-L2, V9    -   SEQ ID NO: 73 light chain CDR-L3, V9    -   SEQ ID NO: 74 heavy chain variable domain VH, V9    -   SEQ ID NO: 75 light chain variable domain VL, V9

Bispecific Anti-HLA-G/Anti-CD3 T Cell Bispecific (TCB) Antibodies:

P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035-093 (P035)):

-   -   SEQ ID NO: 76 light chain 1 P1AF7977    -   SEQ ID NO: 77 light chain 2 P1AF7977    -   SEQ ID NO: 78 heavy chain 1 P1AF7977    -   SEQ ID NO: 79 heavy chain 2 P1AF7977

P1AF7978 (HLA-G-0090-VL-S32P/CD3 Clone 22 (C122)):

-   -   SEQ ID NO: 80 light chain 1 P1AF7978    -   SEQ ID NO: 81 light chain 2 P1AF7978    -   SEQ ID NO: 82 heavy chain 1 P1AF7978    -   SEQ ID NO: 83 heavy chain 2 P1AF7978

P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9):

-   -   SEQ ID NO: 84 light chain 1 P1AF7979    -   SEQ ID NO: 85 light chain 2 P1AF7979    -   SEQ ID NO: 86 heavy chain 1 P1AF7979    -   SEQ ID NO:87 heavy chain 2 P1AF7979

Further Sequences

-   -   SEQ ID NO: 88 exemplary human CD3    -   SEQ ID NO: 89 exemplary cynomolgus CD3    -   SEQ ID NO: 90 Human CD3 epsilon stalk-Fc(knob)-Avi    -   SEQ ID NO: 91 Human CD3 delta stalk-Fc (hole)-Avi    -   SEQ ID NO: 92 CD3_(orig) VH    -   SEQ ID NO: 93 CD3_(orig) VL    -   SEQ ID NO: 94 CD3_(orig) IgG HC    -   SEQ ID NO: 95 P035 IgG HC    -   SEQ ID NO: 96 CD3_(orig)/P035 IgG LC

The Amino Acid Sequences of HLA-G-0090 Antibody (Variable Regions withUnderlined Complementarity Determining Regions (CDRs) and UnmodifiedN-Glycosylation Site in CDR-L1 (bold)):

SEQ ID NO: 7: heavy chain variable domain VH, HLA-G-0090:QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVS S SEQ ID NO: 8:light chain variable domain VL, HLA-G-0090 DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

The Amino Acid Sequences of Modified HLA-G-0090 Antibody Light ChainVariable Regions (with Underlined Complementarity Determining Regions(CDRs) and Modified N-Glycosylation Site in CDR-L1 (Bold)):

SEQ ID NO:10: light chain variable domain VL, HLA-G-0090-N31DDIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 12:light chain variable domain VL, HLA-G-0090-N31L DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 14:light chain variable domain VL, HLA-G-0090-N31Q DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 16:light chain variable domain VL, HLA-G-0090-N315 DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 18:light chain variable domain VL, HLA-G-0090-N31T DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 20:light chain variable domain VL, HLA-G-0090-N31Y DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 22:light chain variable domain VL, HLA-G-0090-N31Y-N38YDIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 24:light chain variable domain VL, HLA-G-0090-532P DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 26:light chain variable domain VL, HLA-G-0090-533A DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 28:light chain variable domain VL, HLA-G-0090-533D DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 30:light chain variable domain VL, HLA-G-0090-533P DIVMTQSPDSLAVSLGERATINC

W YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

The Amino Acid Sequences of Anti-CD3 Binding Moieties (Variable Regionswith Underlined Complementarity Determining Regions (CDRs):

heavy chain variable domain VH, P035-093 (abbreviates as P035)SEQ ID NO: 58 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPASYVSYFAYWGQGTLVTV SSlight chain variable domain VL, P035-093 (P035) SEQ ID NO: 59QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVLheavy chain variable domain VH, Clone 22 (abbreviated as C122)SEQ ID NO: 66 EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTV SSlight chain variable domain VL, Clone 22 (C122) SEQ ID NO: 67QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVLheavy chain variable domain VH, V9 SEQ ID NO: 74EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSlight chain variable domain VL, V9 SEQ ID NO: 75DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQGNTLPWTFGQGTKVEIK

The Amino Acid Sequences of Bispecific Anti-HLA-G/Anti-CD3 T CellBispecific (TCB) Antibodies:

P1AF7977 (HLA-G-0090-VL-S32P/ CD3 P035-093 (P035)):light chain 1 P1AF7977 SEQ ID NO: 76EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNW VRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPA SYVSYFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 2 P1AF7977 SEQ ID NO: 77DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNN KNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFG QGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC heavy chain 1 P1AF7977 SEQ ID NO: 78QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP heavy chain 2 P1AF7977 SEQ ID NO: 79QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSL TVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGA QPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPP1AF7978 (HLA-G-0090-VL-S32P/ CD3 Clone 22 (C122)):light chain 1 P1AF7978 SEQ ID NO: 80 EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRF TISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEClight chain 2 P1AF7978 SEQ ID NO: 81 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC heavy chain 1 P1AF7978SEQ ID NO: 82 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPheavy chain 2 P1AF7978 SEQ ID NO: 83 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9): light chain 1 P1AF7979SEQ ID NO: 84 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTI SVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEClight chain 2 P1AF7979 SEQ ID NO: 85 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC heavy chain 1 P1AF7979SEQ ID NO: 86 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPheavy chain 2 P1AF7979 SEQ ID NO: 87 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDEKVEPKSCDGGGGSGGGGGDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

In the Following Specific Embodiments of the Invention are Listed

1. An antibody that binds to human HLA-G comprising

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6.

2. The antibody according to embodiment 1, wherein the antibody

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

3. The antibody according to any one of embodiments 1 or 2, wherein theantibody comprises a Fc domain of human origin, particularly of the IgGisotype, more particularly of the IgG1 isotype.

4. The antibody according to any one of embodiments 1 or 2, wherein theantibody comprises a constant region of human origin, particularly ofthe IgG isotype, more particularly of the IgG1 isotype, comprising ahuman CH1, CH2, CH3 and/or CL domain.

5. The antibody according to any one of embodiment 1 to 4, wherein theantibody

a) has improved binding properties with respect to maximal binding(Rmax) and/or binding affinity (KD) compared to the (parental) antibodythat comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8(as shown in Example 2).

b) does not crossreact with a modified human HLA-G β2M MHC I complex,wherein the HLA-G specific amino acids have been replaced by HLA-Aconsensus amino acids, the complex comprising SEQ ID NO:40 (as shown inExample 2); and/or

c) does not crossreact with a mouse H2Kd β2M MHC I complex comprisingSEQ ID NO:41(as shown in Example 2); and/or

d) does not crossreact with rat RT1A β2M MHC I complex comprising SEQ IDNO:43(as shown in Example 2).

6. The antibody according to any one of embodiments 1 to 3, wherein theantibody

a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells (ATCC No.HTB36) (as shown in Example 5); or

b) binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36), andinhibits ILT2 binding to (HLA-G expressed on) JEG-3 cells (ATCC No.HTB36) (as shown in Example 5).

7. The antibody according to any one of embodiments 1 to 4, wherein theantibody is a multispecific antibody (preferably a bispecific antibody).

8. The antibody according to embodiment 7, wherein the antibody is abispecific antibody that binds to human HLA-G and to human CD3.

9. The antibody according to embodiment 7, wherein the antibody is abispecific antibody that binds to human HLA-G and to human CD3,comprising a first antigen binding moiety that binds to human HLA-G anda second antigen binding moiety that binds to human CD3, wherein thefirst antigen binding moiety that binds to human HLA-G comprises

A) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:25; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:6;

and wherein the second antigen binding moiety that binds to a T cellactivating antigen binds to human CD3 comprises

C) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:52, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:53, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:54; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:55; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:56 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:57, or

D) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:60, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:61, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:62; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:63; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:64 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:65, or

E) (a) a VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO:68, (ii) CDR-H2 comprising the amino acid sequenceof SEQ ID NO:69, and (iii) CDR-H3 comprising the amino acid sequence ofSEQ ID NO:70; and (b) a VL domain comprising (i) CDR-L1 comprising theamino acid sequence of SEQ ID NO:71; (ii) CDR-L2 comprising the aminoacid sequence of SEQ ID NO:72 and (iii) CDR-L3 comprising the amino acidsequence of SEQ ID NO:73.

10. The bispecific antibody according to embodiment 9,

wherein the first antigen binding moiety

A) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24;or

B) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26,

and wherein the second antigen binding moiety

C) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:58 and a VL domain comprising the amino acid sequence of SEQ IDNO:59; or

D) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:66 and a VL domain comprising the amino acid sequence of SEQ IDNO:67; or

E) comprises a VH domain comprising the amino acid sequence of SEQ IDNO:74 and a VL domain comprising the amino acid sequence of SEQ IDNO:75.

11. The bispecific antibody according to embodiment 10,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:58and a VL domain comprising the amino acid sequence of SEQ ID NO:59.

12. The bispecific antibody according to embodiment 108,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:66and a VL domain comprising the amino acid sequence of SEQ ID NO:67.

13. The bispecific antibody according to embodiment 10,

wherein the first antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7and a VL domain comprising the amino acid sequence of SEQ ID NO:24;

and wherein the second antigen binding moiety

comprises a VH domain comprising the amino acid sequence of SEQ ID NO:74and a VL domain comprising the amino acid sequence of SEQ ID NO:75.

14. The bispecific antibody according to any one of embodiments 8 to 13,wherein

the bispecific antibody shows

a) inhibition of ILT2 and/or ILT4 binding to HLA-G (as shown in Example13); and/or

b) antibody mediated IFN gamma secretion by T cells on SKOV3 cellstransfected with recombinant HLA-G (SKOV3 HLA-G) and/or on JEG3 cellsexpressing endogenous HLA-G wherein the IFN gamma secretion was detected(by Luminex technology) (as shown in Example 14); and or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cellstransfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3 cellsexpressing endogenous HLA-G wherein the cytotoxicity was detected bymeasuring Caspase 8 activation in cells after treatment with bispecificantibody (as shown in Example 15); and/or

-   -   d) in vivo anti-tumor efficacy/tumor regression in humanized NSG        mice bearing SKOV3 human ovarian carcinoma transfected with        recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (as shown in        Example 16); and/or

e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell bi-specific inhumanized NSG mice bearing human breast cancer PDX tumors (BC004) (asshown in Example 17).

15. The bispecific antibody of any one of embodiments 9 to 14, whereinthe first and the second antigen binding moiety is a Fab molecule.

16. The bispecific antibody of any one of embodiments 9 to 15, whereinthe second antigen binding moiety is a Fab molecule wherein the variabledomains VL and VH or the constant domains CL and CH1, particularly thevariable domains VL and VH, of the Fab light chain and the Fab heavychain are replaced by each other.

17. The bispecific antibody of any one of embodiments 9 to 16, whereinthe first antigen binding moiety is a Fab molecule wherein in theconstant domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 the amino acid atposition 147 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index) and the aminoacid at position 213 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index).

18. The bispecific antibody of any one of embodiments 9 to 17,comprising a third antigen binding moiety. wherein the third antigenmoiety is identical to the first antigen binding moiety.

19. The bispecific antibody of any one of embodiments 9 to 18,comprising an Fc domain composed of a first and a second subunit.

20. The bispecific antibody of embodiment 19 wherein the first, thesecond and, where present, the third antigen binding moiety are each aFab molecule; and wherein either (i) the second antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding moiety and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain, or (ii) thefirst antigen binding moiety is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety and the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain; and wherein the third antigen binding moiety, wherepresent, is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain.

21. The bispecific antibody of embodiment 19 or 20, wherein the Fcdomain is a human IgG Fc domain, particularly of the IgG1 isotype.

22. The bispecific antibody of any one of embodiments 19 or 20, whereinthe Fc domain comprises one or more amino acid substitution that reducesbinding to an Fc receptor and/or effector function.

23. The bispecific antibody according embodiment 22, wherein theantibody is of the IgG1 isotype with mutations L234A, L235A and P329G(numbering according to the EU index of Kabat).

24. The bispecific antibody of any one of embodiments 19 to 23, whereinan amino acid residue in the CH3 domain of the first subunit of the Fcdomain is replaced with an amino acid residue having a larger side chainvolume, thereby generating a protuberance within the CH3 domain of thefirst subunit which is positionable in a cavity within the CH3 domain ofthe second subunit, and an amino acid residue in the CH3 domain of thesecond subunit of the Fc domain is replaced with an amino acid residuehaving a smaller side chain volume, thereby generating a cavity withinthe CH3 domain of the second subunit within which the protuberancewithin the CH3 domain of the first subunit is positionable.

25. The bispecific antibody according embodiment 24, wherein theantibody is of IgG1 isotype with mutation T366W in the first subunit ofthe Fc domain and with mutations Y407V, T366S and L368A in the secondsubunit of the Fc domain (numberings according to Kabat EU index).

26. The bispecific antibody according embodiment 25, wherein theantibody comprises an additional mutation S354C in the first subunit ofthe Fc domain and an additional mutation Y349C in the second subunit ofthe Fc domain (numberings according to Kabat EU index).

27. The bispecific antibody according embodiment 25, wherein theantibody comprises an additional mutation Y349C in the first subunit ofthe Fc domain and an additional S354C mutation in the second subunit ofthe Fc domain (numberings according to Kabat EU index).

28. Isolated nucleic acid encoding the antibody according to any one ofembodiments 1-4 or the bispecific antibody according to any one ofembodiments 9-27.

29. A host cell, preferably an eukaryotic host cell, comprising thenucleic acid of embodiment 28.

30. A method of producing the antibody according to any one ofembodiments 1-4 or the bispecific antibody according to any one ofembodiments 9-227 comprising culturing the host cell of embodiment 29 sothat the antibody or bispecific antibody is produced.

31. The method of embodiment 30, further comprising recovering theantibody or bispecific antibody from the host cell.

32. The antibody according to any one of embodiments 1-4 or thebispecific antibody according to any one of embodiments 9-27, whereinthe antibody is produced according to a method of embodiments 30 to 31and wherein the host cell is an eukaryotic host cell (in one preferredembodiment a mammalian host cell, in another preferred embodiment a CHOcell).

33. The antibody according to any one of embodiments 1-4 or thebispecific antibody according to any one of embodiments 9-27, whereinthe antibody is produced in an eukaryotic host cell (in one preferredembodiment a mammalian host cell, in another preferred embodiment a CHOcell).

34. The antibody according to any one of embodiments 1-4 or thebispecific antibody according to any one of embodiments 9-27 for use asa medicament.

35. The antibody according to any one of embodiments 1-4 or thebispecific antibody according to any one of embodiments 9-27 for use intreating cancer.

36. Use of the antibody according to any one of embodiments 1-4 or thebispecific antibody according to any one of embodiments 9-27 in themanufacture of a medicament.

37. The use of embodiment 36, wherein the medicament is for treatment ofcancer.

38. A method of treating an individual having cancer comprisingadministering to the individual an effective amount of the antibodyaccording to any one of embodiments 1-4 or the bispecific antibodyaccording to any one of embodiments 9-27.

EXAMPLES

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany) The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany)

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. full length antibodyheavy chain, full length antibody light chain, or an MHC class Imolecule, e.g. HLA-G, or an MHC class I molecule fused to peptide andbeta-2 microglobulin, e.g. HLA-G fused to HLA-G binding peptide and orbeta-2 microglobulin) a transcription unit comprising the followingfunctional elements is used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain or MHC class I molecule), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to beexpressed the basic/standard mammalian expression plasmid contains

-   -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

Protein Determination

The protein concentration of purified polypeptides was determined bydetermining the optical density (OD) at 280 nm, using the molarextinction coefficient calculated on the basis of the amino acidsequence of the polypeptide.

Example 1

Generation of HLA-G Chimeric Molecules

Due to high homology (>98%) with other MHC I molecules, immunisationwith HLA-G molecules results in generation of polyclonal sera, composedof a mixture of MHC-I crossreactive antibodies as well as truly HLA-Gspecific antibodies.

So far no tools have been provided to select truly HLA-G specificantibodies without crossreactivity to other human MHC-I (e.g. HLA-A),and to further select those with receptor blocking function.

We identified unique HLA-G positions in combination to positionsnecessary for structural conformity and receptor interaction (ILT2/4 andKIR2DL4.)

Unique and proximal positions of human HLA-G were then “grafted” on MHCclass I complex molecules from different rodent species (such as ratRT1A and mouse H2kd) to generate “chimeric” immunogen/screeningantigens.

Antibodies generated were subjected to stringent screening/testing forbinding/specificity, (and no crossreactivity/no specificity tocounterantigens, respectively)

antigens for binding testing:

-   -   rec. HLA-G expressed as human HLA-G β2M MHC complex comprising        SEQ ID NO: 43    -   HLA-G specific sequences grafted onto rat RT-1 and mouse H2kd        (SEQ ID NO: 46: human HLA-G/mouse H2Kd β2M MHC class I complex        wherein the positions specific for human HLA-G are grafted onto        the mouse H2Kd framework and SEQ ID NO: 48: human HLA-G/rat RT1A        β2M MHC class I complex wherein the positions specific for human        HLA-G are grafted onto the rat RT1A framework)    -   Natural HLA-G MHC class I complex expressing cells (e.g. Jeg3        cells), or human HLA-G transfected cell lines SKOV3 HLA-G+ and        PA-TU-8902 HLA-G+

counter antigens for crossreactivity testing:

-   -   Counter antigens (MHC class I complexes) with other HLA-A        sequences (HLA-A2 and        HLA-G^(degrafted with H1A-A consensus sequence)) combined with        different peptides) (see e.g. SEQ ID NO 35 (HLA-A2) and SEQ ID        NO: 40 HLA-A consensus sequence on HLA-G framework)    -   Counter antigens (MHC class I complexes) from other species such        as rat RT-1 and mouse H2kd (SEQ ID NO: 43 and SEQ ID NO: 41)    -   Unmodified tumor cell lines SKOV3 and PA-TU-8902, which are        characterized by absence of HLA-G expression.

Design of Chimeric HLA-G Antigens to Determine the Specific Binding ofAnti-HLA-G Antibodies (see FIG. 2):

Design of a chimeric rat MHC I molecule (RT1-A) carrying HLA-G uniquepositions (SEQ ID NO: 44) for use in for use in binding assays:

HLA-G unique positions were identified by the alignment of 2579 HLA-A,3283 HLA-B, 2133 HLA-C, 15 HLA-E, 22 HLA-F, and 50 HLA-G sequences fromIMGT (as available on 6 Feb. 2014). Those residues of HLA-G that occurin less than 1% (mostly ˜0%) of the sequences of any of the 3 sequencesets HLA-A, HLA-B, and a combined set of HLA-C+HLA-E+HLA-F are calledHLA-G unique positions.

The 4 core HLA-G unique positions (2 in alpha-1 and 2 in alpha-3) showno polymorphism in the set of HLA-G sequences and none of the other HLAgenes contain the HLA-G specific residues at these positions (except 1×HLA-A for M100, 1× HLA-B for Q103, and 1× HLA-C for Q103).

The crystal structure of rat RT1-A (Rudolph, M. G. et al. J. Mol. Biol.324: 975-990 (2002); PDB code: 1KJM) was superimposed on the crystalstructure of human HLA-G (Clements, C. S. et al. PROC. NATL. ACAD. SCI.USA 102: 3360-3365 (2005); PDB code: 1YDP). The overall structure of thealpha-chain and the associated beta-2-microglobulin is conserved.

HLA-G unique positions were identified in the RT1-A structure bycomparison of the sequence and structural alignments. In a first step,unique HLA-G positions were identified that are exposed on the molecularsurface of HLA-G and RT1-A and thus accessible for an antibody. Uniquepositions that are buried within the protein fold were excluded forengineering. In a second step, structurally proximal residues wereidentified, that also need to be exchanged to make the correspondingregion “HLA-G-like”, i.e. to generate real HLA-G epitopes containing theunique positions rather than generating HLA-G/rat RT1-A chimericepitopes that would be artificial. All the positions that were thusselected for mutation were analyzed for structural fit of the respectiveresidue from HLA-G to avoid possible local disturbances of the molecularstructure upon mutation.

A chimeric mouse MHC I molecule (H2Kd) carrying HLA-G unique positions(SEQ ID NO: 42) for use in binding assays was generated analogously.

Design of HLA-A Based Counter Antigens by “De-Grafting” of HLA-G UniquePositions Towards a HLA-A Consensus Sequence for Crossreactivity Testing(SEQ ID NO:40=Modified Human HLA-G 112M MHC Class I Complex (wherein theHLA-G Specific Amino Acids Have Been Replaced by HLA-A Consensus AminoAcids (=Degrafted HLA-G))

Unique positions derived from the multiple sequence alignment wereanalyzed in a crystal structure of human HLA-G (PDB code: 1YDP). First,positions that are not exposed on the HLA-G surface and are thus notaccessible for an antibody were excluded for engineering. Second, thesurface exposed residues were analyzed for feasibility of amino acidexchange (i.e. exclusion of possible local disturbances of the molecularstructure upon mutation of the relevant position). In total, 14positions were validated for exchange. The amino acids in the validatedpositions were mutated towards a HLA-A consensus sequence derived from amultiple sequence alignment of 2579 HLA-A sequences downloaded from IMGT(as available on 6 Feb. 2014).

Generation of Expression Plasmids for Soluble Classical andNon-Classical MHC Class I Molecules

The recombinant MHC class I genes encode N-terminally extended fusionmolecules consisting of a peptide know to be bound by the respective MHCclass I molecule, beta-2 microglobulin, and the respective MHC class Imolecule.

The expression plasmids for the transient expression of soluble MHCclass I molecules comprised besides the soluble MHC class I moleculeexpression cassette an origin of replication from the vector pUC18,which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the soluble MHC class I molecule comprised thefollowing functional elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   an N-terminally truncated S. aureus sortase A encoding nucleic        acid, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

The amino acid sequences of the mature soluble MHC class I moleculesderived from the various species are:

SEQ ID NO: 39: exemplary human HLA-G β2M MHC class I complex

SEQ ID NO: 40: exemplary modified human HLA-G β2M MHC class I complex(wherein the HLA-G specific amino acids have been replaced by HLAconsensus amino acids (=degrafted HLA-G see also FIG. 2)

SEQ ID NO: 41: exemplary mouse H2Kd β2M MHC class I complex

SEQ ID NO: 42: exemplary human HLA-G/mouse H2Kd β2M MHC complex whereinthe positions specific for human HLA-G are grafted onto the mouse H2Kdframework

SEQ ID NO: 43: exemplary rat RT1A β2M MHC class I complex

SEQ ID NO: 44: exemplary human HLA-G/rat RT1A β2M MHC complex whereinthe positions specific for human HLA-G are grafted onto the rat RT1Aframework

For the exemplary HLA-A2 132M MHC class I complex the followingcomponents were used and the complex was expressed in E. coli andpurified.

MHCI complex HLA-A2/b2M (SEQ ID NOs 35 and 33) (both with an additionalN-terminal methionine) +VLDFAPPGA peptide (SEQ ID NO: 46) +linker andhis-Tag (SEQ ID NO: 45)

Example 2

Removal of N-glycosylation Motif in CDR-L1

The CDR-L1 of anti-HLA-G antibody HLA-G-0090 contains a classicalN-glycosylation motif “NSS” comprising positions 31 to 33 of the lightchain (LC). It was decided to remove this motif as it could constitute apotential developability liability. An homology model of the variableregion of HLA-G-0090 indicated that LC positions 31 to 33 are highlysolvent accessible. Furthermore, the side chains of N31 and S32 arepredicted to point inwards, in the direction of CDR-H3, making themlikely candidates for being part of the antibody paratope. In fact, anumber of published antibody-antigen X-ray complex structures documentthese residues to be undergoing chemical interactions with the antigen.Therefore, the risk of worsening the binding affinity of the antibody byintroducing mutations at LC positions 31-33 was considered high. Toameliorate the risk, 11 different variants of antibody HLA-G-0090 withmutations on LC positions 31, 32, and 33 were designed and produced inHEK293F cells in an IgG1 format.

Note that mutant variant HLA-G-0090-VL-N31Y-N38Y contained a secondmutation (N38Y), apart from the N-glycosylation motif, and not relatedto its removal, to increase germline identity.

Summary of Anti-HLA-G Antibody Sequences (SEQ ID NOs of Variable Regionsand CDRs):

CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 VH VL SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID Anti-HLA-G antibody NO: NO: NO: NO: NO:NO: NO: NO: HLA-G-0090 1 2 3  4 5 6 7  8 HLA-G-0090-VL- 1 2 3  9 5 6 710 N31D HLA-G-0090-VL- 1 2 3 11 5 6 7 12 N31L HLA-G-0090-VL- 1 2 3 13 56 7 14 N31Q HLA-G-0090-VL- 1 2 3 15 5 6 7 16 N31S HLA-G-0090-VL- 1 2 317 5 6 7 18 N31T HLA-G-0090-VL- 1 2 3 19 5 6 7 20 N31Y HLA-G-0090-VL- 12 3 21 5 6 7 22 N31Y-N38Y HLA-G-0090-VL- 1 2 3 23 5 6 7 24 S32PHLA-G-0090-VL- 1 2 3 25 5 6 7 26 S33A HLA-G-0090-VL- 1 2 3 27 5 6 7 28S33D HLA-G-0090-VL- 1 2 3 29 5 6 7 30 S33P

Binding and other properties of the obtained anti-HLA-G specificantibodies and biological activities were determined as described in thefollowing Examples, and compared to the known reference, HLA-G-0090.

Expression and Purification

The expression yields from a 0.5 L expression in HEK293F cells afterpurification by affinity chromatography (MabSelect Sure) and dialysisare shown in the following table.

Monomer Content [%] Purity [%] mg (analytical SEC) (Caliper) HLA-G-00903.2 98 99 HLA-G-0090-VL- 0.4 97 98 N31D HLA-G-0090-VL- 0.4 98 99 N31LHLA-G-0090-VL- 0.4 91 95 N31Q HLA-G-0090-VL- 0.3 93 98 N31SHLA-G-0090-VL- 0.4 85 89 N31T HLA-G-0090-VL- 0.3 89 94 N31YHLA-G-0090-VL- 0.7 94 98 N31Y-N38Y HLA-G-0090-VL- 2.4 98 99 S32PHLA-G-0090-VL- 2.1 98 99 S33A HLA-G-0090-VL- 2.4 98 99 S33DHLA-G-0090-VL- 1.4 98 99 S33P

Variants with mutations involving position LC 31 (N31X) showed stronglydecreased expression titers and, often, impaired material quality, whileLC 32 and LC33 positions variants showed good to acceptable expressiontiters and good material quality.

HLA-G Binding

wt-HLA-G Affinity/Kinetic

Affinity to wt-HLA-G complex (SEQ ID NO: 39) of individual 5 nManti-HLA-G antibodies was determined by capturing with anti-hFc (GEHealthcare BR-1008-39) on a CM3 sensor chip and the injection ofwt-HLA-G antigen at a concentration of 11 nM to 300 nM diluted in HBS-P+(GE Healthcare) running buffer and a flow rate of 60 μl/min with 120 sassociation time and 600 s dissociation time. After each cycle thesurface was regenerated by washing with 3M MgCl2 The kinetics bindingcurves were evaluated using T200 evaluation software and for thecalculation of binding properties 1:1 Langmuir binding model was used.

RAC of HLAG Antibodies (Relative Active Concentration) (Assay Scheme isShown in FIG. 3)

RAC of HLA-G binders of individual anti-HLA-G antibodies (10 nMsolutions in HBS-P+) was determined by capturing with anti-hFc (GEHealthcare BR-1008-39) on a CM3 sensor chip and the injection ofwt-HLA-G antigen at a concentration of 300 nM diluted in HBS-P+ (GEHealthcare) running buffer and a flow rate of 10 μl/min with 60 sassociation time and 600 s dissociation time. After each cycle thesurface was regenerated by washing with 3M MgCl2. Final RAC and Rmaxvalues are calculated from “binding” report points and the capturinglevels. Rmax=(MW analyte/MW ligand)*RU capturing ligand*stoichiometry ofinteraction.

The 11 variants were evaluated in an SPR binding experiment in which theantibodies were immobilized on a CM3 chip via the Fc part andrecombinant single-chain HLA-G monomer/human HLA-G β2M MHC class Icomplex (SEQ ID NO: 39) was used as the analyte. The kinetic parameterswere determined by a single cycle kinetic measurement on a Biacore T200device at 25° C.

ka kd t½ Kd Rmax [1/ms] [1/s] [s] [nM] [%] HLA-G-0090 1.19E+06 1.38E−03501 1.2 86 HLA-G-0090- 1.94E+05 3.91E−03 177.3 20.2 66 VL-N31DHLA-G-0090- 5.28E+06 8.71E−03 79.6 1.7 91 VL-N31L HLA-G-0090- 1.91E+063.73E−03 185.9 2.0 91 VL-N31Q HLA-G-0090- 3.83E+05 9.22E−04 751.8 2.4 77VL-N31S HLA-G-0090- 3.42E+05 7.69E−04 901.9 2.3 75 VL-N31T HLA-G-0090-5.49E+05 1.07E−03 646.2 2.0 78 VL-N31Y HLA-G-0090- 1.82E+09 4.30E+01 023.6 57 VL-N31Y- N38Y-GL HLA-G-0090- 1.19E+06 1.24E−03 557.7 1.0 97VL-S32P HLA-G-0090- 1.24E+06 1.24E−03 558.2 1.0 98 VL-S33A HLA-G-0090-7.17E+05 3.44E−03 201.7 4.8 95 VL-S33D HLA-G-0090- 1.34E+06 2.36E−03294.3 1.8 94 VL-S33P

Among those, the four variants with acceptable expression titers(HLA-G-0090-VL-S32P, HLA-G-0090-VL-S33A, HLA-G-0090-VL-S33D,HLA-G-0090-VL-S33P) were evaluated further in a more accurate multicycle kinetics measurement using the same SPR device and experimentalsetup.

ka kd t½ Kd Rmax [1/ms] [1/s] [s] [nM] [%] HLA-G-0090 1.30E+06 1.78E−03389.9 1.4 87 HLA-G-0090- 1.27E+06 1.57E−03 440.6 1.2 99 VL-S32PHLA-G-0090- 1.24E+06 1.49E−03 465.9 1.2 99 VL-S33A HLA-G-0090- 8.97E+056.67E−03 103.9 7.4 98 VL-S33D HLA-G-0090- 1.36E+06 3.68E−03 188.1 2.7 97VL-S33P

The two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A have animproved binding affinity compared to the parental antibody HLA-G-0090while the two variants HLA-G-0090-VL-S33D and HLA-G-0090-VL-S33P arelosing binding affinity by a factor of 5 and 2, respectively.Surprisingly, for the tested variants, the removal of theN-glycosylation motif leads to a higher Rmax value in the kineticsmeasurement, indicating a higher fraction of successful complexformation than for the N-glycosylated antibody.

Crossreactivity of Anti HLA-G Antibodies and Variants to Soluble HumanHLA-G, Soluble Degrafted Human HLA-G with HLA-A Consensus SpecificSequence, and Rat/Mouse Homologues

To further investigate the binding properties of the twobinding-affinity improved variants HLA-G-0090-VL-S32P andHLA-G-0090-VL-S33A, SPR binding experiments to four counter-screeningconstructs were performed. These recombinant single-chain peptide-MHCcomplex constructs were murine H2-K1 (SEQ ID NO: 41), rat RT1 (SEQ IDNO: 43), and human HLA-G β2M MHC class I complex, wherein the HLA-Gspecific amino acids have been replaced by HLA-A consensus amino acids(SEQ ID NO:40). The latter construct constitutes a version of HLA-G inwhich all HLA-G specific residues have been replaced by their HLA-Aconsensus counterparts. Again, the antibodies were immobilized on CM3chip and the single-chain peptide-MHC constructs were used as analyte ona Biacore T200 device at 25° C.

Antigen Antibody Interaction murine H2-K1 (SEQ HLA-G-0090 no bindinginteraction ID NO: 41) murine H2-K1 (SEQ HLA-G-0090-VL-S32P no bindinginteraction ID NO: 41) murine H2-K1 (SEQ HLA-G-0090-VL-S33A no bindinginteraction ID NO: 41) rat RT1 (SEQ HLA-G-0090 no binding interaction IDNO: 43) rat RT1 (SEQ HLA-G-0090-VL-S32P no binding interaction ID NO:43) rat RT1 (SEQ HLA-G-0090-VL-S33A no binding interaction ID NO: 43)HLA-A consensus on HLA-G-0090 no binding interaction HLA-G frame (SEQ IDNO: 40) HLA-A consensus on HLA-G-0090-VL-S32P no binding interactionHLA-G frame (SEQ ID NO: 40) HLA-A consensus on HLA-G-0090-VL-S33A nobinding interaction HLA-G frame (SEQ ID NO: 40) HLA-G (SEQ HLA-G-0090very strong binding ID NO: 39) interaction HLA-G (SEQ HLA-G-0090-VL-S32Pvery strong binding ID NO: 39) interaction HLA-G (SEQ HLA-G-0090-VL-S33Avery strong binding ID NO: 39) interaction

Stability Under Stress

The parental antibody as well as the two derived variantsHLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A were stressed for 13 daysunder two different conditions:

-   -   pH 6.0 20 mM His/HisCl, 140 mM NaCl; at 40° C. (His 40° C.)    -   pH 7.4 PBS; at 37° C. (PBS 37° C.).

Afterwards, the material was analysed using SEC, and SPR to investigatechemical degradation and possible effects on target binding. Forreference, the stressed material was compared with material kept understorage conditions:

-   -   pH 6.0 20 mM His/HisCl, 140 mM NaCl; frozen at −80° C. (Ref.)

The results are listed in the following table (relative in % compared toRef):

HLA-G- HLA-G- 0090_VL- 0090_VL- HLA-G- Parameter S32P S33A 0090 SECmonomer Ref. 100 99 99 [%] His 40° C. 98 98 98 PBS 37° C. 98 98 98relative HLA-G Ref. 100 100 100 binding signal His 40° C. 99 99 99compared to Ref PBS 37° C. 98 96 96 (as 100%) ± stress, by SPR RAC

While all three antibodies are showing a very similar stability profile,variant HLAG-0090_VL-S32P is retaining more HLA-G binding (SPR relativeactive concentration (RAC)) after stress in PBS at 37° C. than the othertwo, including the parental antibody.

Thermal Stability Testing

For thermal stability testing of the purified proteins the Uncle devicewas used (UNCHAINED LABS, Boston, Mass., USA). Static light scatteringat 266 nm and 473 nm and in parallel intrinsic fluorescence is herebyused to determine aggregation temperature (Tagg) and melting temperature(Tm) of the purified proteins. A temperature ramp from 30° C. to 90° C.in 0.1° C./min steps was run. Glass cuvettes with 9 μl volume persamples were used and the concentration was 1 mg/mL in 20 mM Histidin,140 mM NaCl, pH 6.0 buffer. For analysis, the software UNcle analysis(UNCHAINED LABS) was used.

Mass Spectrometry Analysis and N-Glcyosylation

The deconvoluted mass spectra of the intact samples are documenting theimpact on the N-Glycosylation of the removal of the N-glycosylationsite/NSS motif in HLA-G-0090_VL-S32P and HLA-G-0090_VL-S33A. The sampleswere prepared with PNGase F to remove all N-linked glycans and obtainthe molecular mass of the antibody only. While PNGase F is fullyspecific for cleavage of N-linked Fc-glycans, it shows much lessefficacy when cleaving N-linked Fab-glycans. HLA-G-0090 is showing aclear N-glycosylation pattern coming from incomplete deglycosylation andtherefore indicating Fab-glycosylation ((see FIG. 4A). No signs ofresidual N-glycosylation can be detected for HLA-G-0090_VL-S32P andHLA-G-0090_VL-S33A (see FIG. 4B).

Example 3

ILT2 and -4 Binding Inhibition of Anti-HLA-G Antibodies

The ELISA is set up by coating the Fc tagged ILT2 and ILT4 respectivelyto Maxisorp microtiter plates. After incubation and washing steps, therespective antibodies are added at a concentration of 100 nM. SolubleHis tagged monomeric, dimeric or trimeric HLA-G was added to the wells.After incubation and washing steps, detection of bound receptor iscarried out by anti-His-antibody-POD conjugates. Percentage inhibition(%) is calculated in comparison to values obtained from wells withILT2/4+HLA-G (mono-, di-, or Trimer) without anti HLA-G or ILT2/4antibodies (100% binding=0% inhibition) and shown in a table

Example 4

Binding of HLA-G Antibodies to Natural or Recombinant HLA-G Expressed onCells (as Assessed by FACS Analysis)

For flow cytometry analysis, cells were stained with anti HLA-G mAbs at4° C. Briefly, each cell suspension was transferred into a polypropylenetube (2×10⁵ cells/tube) and prechilled at 5° C. for 10 minutes. Cellswere then washed with 2 ml FACS Buffer (4° C.) and centrifuged at 300 gfor 5 minutes. Anti-HLA-G antibodies HLAG-0090-VL-S32P,HLAG-0090-VL-S33A, HLAG-0090 were diluted in staining buffer to astarting concentration of 50 μg/ml. A 5-fold serial dilution of theantibodies was performed to get the final concentrations (10 μg/ml, 2μg/ml, 0.4 μg/ml, 0.08 μg/ml, 0.016 μg/ml, 0.0032 μg/ml). FACS bufferwas then aspirated from the tubes and the cell pellets were resuspendedin 100 μl of the antibody solution and incubated for 1 h at 5° C. Cellswere then washed once with 2 ml staining buffer and centrifuged at 300 gfor 5 minutes.

For detection fluorescent labeled anti-species antibody (goat anti-humanIgG (H+L) conjugated to Alexa 488, Life technologies #A11013) wasdiluted to 10 μg/ml in a staining buffer and cell pellets wereresuspended in 100 μl of detection antibody. After a 1 hour incubationat 5° C. cells were again washed once with 2 ml of staining buffer,resuspended in 500 μl of staining buffer and measured on a FACS CELESTA

An exemplary FACS staining for anti-HLA-G antibodies HLA-G-0090,HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32P (10 μg/ml) is shown in theFACS overlays of FIG. 5: Both deglycosylated variants of the HLA-G 0090,HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32Pshow good binding to HLA-Gexpressing SKOV3 cells and JEG cells but not to parental SKOV3 cells.The MFI values of the respective HLA-G antibodies are indicated in thehistograms.

Example 5

Anti HLA-G Antibodies Inhibit/Modulate the Binding of ILT2 to HLA-GExpressed on JEG3 Cells

For analysis, JEG3 cells (ATCC HTB36) were stained with ILT2-c-Myc-Fcfusion protein (control=no inhibition) with or without pre-incubationwith different anti-HLA-G antibodies.

Binding/Inhibition of binding was determined as follows: RecombinantILT2-c-Myc-Fc protein was added to JEG3 cells either pre-incubated antiHLA-G mAbs as described or to untreated JEG3 cells as reference. For thepre-incubation with anti-HLA-G antibodies, 2×10⁵ cells were transferredinto a polypropylene tubes. Anti HLA-G antibodies HLAG-0090-VL-S32P,HLAG-0090-VL-S33A and HLA-G-0090 were diluted in staining buffer to aconcentration of 80 μg/ml and 25 μl of the antibody solution was addedto the prepared cells and incubated for 1 h at 5° C. The ILT2-c-Myc-Fcor (control human IgG (Jackson-Immuno-Research #009-000-003)) werediluted in staining buffer to a 2-fold concentration of 20 μg/ml andadded to the prepared cells at a final concentration of 10 μg/ml andincubated for 2 h at 5° C. Cells were washed twice with 200 μl ofstaining buffer. Human ILT2-c-Myc-Fc protein was detected withfluorescent labeled Anti-Myc tag (9E10) Alexa Fluor 647 (abcam;#ab223895) at a dilution of 10 μg/ml in staining buffer. Cells wereresuspended in 50 μl detection antibody dilution and incubated for 1hour at 5° C. Cells were then washed once with 2 ml staining buffer andresuspended in 500 μl of staining buffer before measuring at a FACSCELESTA

As control, the anti-HLA-G antibodies bound to JEG-3 pre-incubated cellswere detected by using anti-species antibody (goat anti-human IgG (H+L)conjugated to Alexa 488, Life technologies #A11013), was diluted to 10μg/ml in staining buffer and cell pellets were resuspended in 100μl/well detection antibody. After a 1 hour incubation at 5° C. cellswere again washed once with staining buffer, resuspended in 500 μl ofstaining buffer and measured at a FACS CELESTA

The histograms in FIG. 6 show the respective ability of the HLA-Gantibodies to modify/inhibit the interaction and binding of recombinantILT2 to HLA-G naturally expressed on JEG3 tumor cells.

The following table summarizes the results from the experiments. Thebinding of the anti-HLA-G antibodies to JEG3 cells is depicted as +=weakbinding −+++=strong binding. The ability of the anti-HLA-G antibodieseither to inhibit/block or increase the binding of ILT2 to the HLA-Gexpressing JEG3 cells. In the last column, the binding of therecombinant ILT2 to the cells or the inhibition/blockade thereof isshown/quantified (staining of ILT2-c-Myc-Fc in the absence of ananti-HLA-G antibody was set to 100% binding=0% inhibition):

Binding to HLA-G:ILT2 Inhibition of ILT2 Antibody JEG-3 cellsinteraction binding to Jeg3 cells ILT2-Fc w/o — —  0% inhibitionAntibody HLA-G-0090 +++ inhibits binding 72% inhibition of ILT2HLAG-0090- +++ inhibits binding 72% inhibition VL-S32P of ILT2HLAG-0090- +++ inhibits binding 72% inhibition VL-S33A of ILT2

Example 6

Generation of Optimized CD3 Binder

Starting from a previously described CD3 binder, termed “CD3_(orig)”herein (see for details e.g. WO2014/131712 incorporated herein byreference) comprising the VH and VL sequences of SEQ ID NOs 92 and 93and we aimed at optimizing properties of this binder by removal of twoasparagine deamidation sequence motifs at Kabat positions 97 and 100 ofthe heavy chain CDR3.

To this aim, we generated an antibody library, suitable for phagedisplay, of the heavy chain with both asparagines at Kabat position 97and 100 removed, and in addition the CDRs H1, H2, and H3 randomized inorder to compensate for loss of affinity caused by replacing Asn97 andAsn100 through an affinity-maturation process.

This library was put on a filamentous phage via fusion to minor coatprotein p3 (Marks et al. (1991) J Mol Biol 222, 581-597) and selectedfor binding to recombinant CD3ε.

10 candidate clones were identified in the initial screening, showingacceptable binding on recombinant antigen as measured by SPR as Fabfragments (produced in E. coli).

Only one of these clones, however, showed acceptable binding activity toCD3 expressing cells as measured by flow cytometry after conversion toIgG format.

The selected clone, termed P035-093 (P035) (=“CD3_(opt)”) herein andcomprising the VH and VL sequences of SEQ ID NOs 58 and 59,respectively, was further evaluated and converted into bispecific formatas described in the following.

Example 7

Binding of Optimized CD3 Binder to CD3

Binding to Recombinant CD3

Binding to recombinant CD3 was determined by surface plasmon resonance(SPR) for the optimized CD3 binder P035-093 (P035) (=“CD3_(opt)”) andthe original CD3 binder “CD3_(orig)”, both in human IgG1 format withP329G L234A L235A (“PGLALA”, EU numbering) mutations in the Fc region(SEQ ID NOs 94 and 96 (CD3_(orig)) and SEQ ID NOs 95 and 96(P035=CD3_(opt))).

In order to assess the effect of the deamidation site removal and itseffect on the stability of the antibodies, binding of the original andthe optimized CD3 binder to recombinant CD3 was tested after temperaturestress for 14 days at 37° C. or 40° C. Samples stored at −80° C. wereused as reference. The reference samples and the samples stressed at 40°C. were in 20 mM His, 140 mM NaCl, pH 6.0, and the samples stressed at37° C. in PBS, pH 7.4, all at a concentration of 1.2-1.3 mg/ml. Afterthe stress period (14 days) samples in PBS were dialyzed back to 20 mMHis, 140 mM NaCl, pH 6.0 for further analysis.

Relative Active Concentration (RAC) of the samples was determined by SPRas follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fabcapturing antibody (GE Healthcare, #28958325) was immobilized on aSeries S Sensor Chip CMS (GE Healthcare) using standard amine couplingchemistry, resulting in a surface density of 4000-6000 resonance units(RU). As running and dilution buffer, HBS-P+(10 mM HEPES, 150 mM NaCl pH7.4, 0.05% Surfactant P20) was used. CD3 antibodies with a concentrationof 2 μg/ml were injected for 60 s at a flow rate of 5 CD3 antigen (seebelow) was injected at a concentration of 10 μg/ml for 120 s anddissociation was monitored at a flow rate of 5 μl/min for 120 s. Thechip surface was regenerated by two consecutive injections of 10 mMglycine pH 2.1 for 60 s each. Bulk refractive index differences werecorrected by subtracting blank injections and by subtracting theresponse obtained from the blank control flow cell. For evaluation, thebinding response was taken 5 seconds after injection end. To normalizethe binding signal, the CD3 binding was divided by the anti-Fab response(the signal (RU) obtained upon capture of the CD3 antibody on theimmobilized anti-Fab antibody). The relative active concentration wascalculated by referencing each temperature stressed sample to thecorresponding, non-stressed sample.

The antigen used was a heterodimer of CD3 delta and CD3 epsilonectodomains fused to a human Fc domain with knob-into-hole modificationsand a C-terminal Avi-tag (see SEQ ID NOs 90 and 91).

The results of this experiment are shown in FIG. 15. As can be seen, theoptimized CD3 binder CD3_(opt) P035-093 (P035) (=CD3_(opt)) showedstrongly improved binding to CD3 after temperature stress (2 weeks at37° C., pH 7.4) as compared to the original CD3 binder CD3_(orig). Thisresult demonstrates that the deamidation site removal was successful,and has yielded an antibody with superior stability properties, relevantfor in vivo half-life, as well as formulation of the antibody at neutralpH.

Binding to CD3 on Jurkat Cells

Binding to CD3 on the human reporter T-cell line Jurkat NFAT wasdetermined by FACS for the optimized CD3 binder P035-093 (P035)(=CD3_(opt)) and the original CD3 binder “CD3_(orig)”, both in humanIgG1 format with P329G L234A L235A (“PGLALA”, EU numbering) mutations inthe Fc region (SEQ ID NOs 94 and 96 (CD3_(orig)) and SEQ ID NOs 95 and96 (P035=CD3_(opt))).

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega#CS176501) are a human acute lymphatic leukemia reporter cell line witha NFAT promoter, expressing human CD3. The cells were cultured inRPMI1640, 2 g/l glucose, 2 g/l NaHCO₃, 10% FCS, 25 mM HEPES, 2 mML-glutamine, 1× NEAA, 1× sodium-pyruvate at 0.1-0.5 mio cells per ml. Afinal concentration of 200 μg per ml hygromycin B was added whenevercells were passaged.

For the binding assay, Jurkat NFAT cells were harvested, washed with PBSand resuspended in FACS buffer. The antibody staining was performed in a96-well round bottom plate. Therefore 100'000 to 200'000 cells wereseeded per well. The plate was centrifuged for 4 min at 400×g and thesupernatant was removed. The test antibodies were diluted in FACS bufferand 20 μl of the antibody solution were added to the cells for 30 min at4° C. To remove unbound antibody, the cells were washed twice with FACSbuffer before addition of the diluted secondary antibody (PE-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific;Jackson ImmunoResearch #109-116-170). After 30 min incubation at 4° C.unbound secondary antibody was washed away. Before measurement the cellswere resuspended in 200 μl FACS buffer and then analyzed by flowcytometry using a BD Canto II device.

As shown in FIG. 16, the optimized CD3 binder P035-093 (P035)(=CD3_(opt)) and the original CD3 binder “CD3_(orig)” bound comparablywell to CD3 on Jurkat cells.

Example 8

Functional Activity of Optimized CD3 Binder

The functional activity of the optimized CD3 binder “CD3_(opt)” wastested in a Jurkat reporter cell assay and compared to the activity ofthe original CD3 binder “CD3_(orig)”. To test the functional activity ofthe IgGs, anti-PGLALA expressing CHO cells were co-incubated with JurkatNFAT reporter cells in the presence of increasing concentrations ofCD3_(opt) human IgG1 PGLALA or CD3_(orig) human IgG1 PGLALA. Activationof CD3 on the Jurkat NFAT reporter cells upon T cell cross-linkinginduces the production of luciferase and luminescence can be measured asan activation marker. CD3_(orig) human IgG1 wt was included as negativecontrol which cannot bind to anti-PGLALA expressing CHO cells andtherefore cannot be crosslinked on Jurkat NFAT cells. A schematicillustration of the assay is provided in FIG. 17.

Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to expresson their surface an antibody that specifically binds human IgG₁Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference). Thesecells were cultured in DMEM/F12 medium containing 5% FCS+1% GluMax. TheJurkat NFAT reporter cells are as described in Example 7.

Upon simultaneous binding of the CD3 huIgG1 PGLALA to anti-PGLALAexpressed on CHO and CD3 expressed on Jurkat-NFAT reporter cells, theNFAT promoter is activated and leads to expression of active fireflyluciferase. The intensity of luminescence signal (obtained upon additionof luciferase substrate) is proportional to the intensity of CD3activation and signaling. Jurkat-NFAT reporter cells grow in suspensionand were cultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO3, 10% FCS, 25mM HEPES, 2 mM L-glutamin, 1× NEAA, 1× sodium-pyruvate at 0.1-0.5 miocells per ml, 200 μg per ml hygromycin. For the assay, CHO cells wereharvested and viability determined using ViCell. 30 000 targetcells/well were plated in a flat-bottom, white-walled 96-well-plate(Greiner bio-one #655098) in 100 μl medium and 50 μl/well of dilutedantibodies or medium (for controls) were added to the CHO cells.Subsequently, Jurkat-NFAT reporter cells were harvested and viabilityassessed using ViCell. Cells were resuspended at 1.2 mio cells/ml incell culture medium without hygromycin B and added to CHO cells at 60000 cells/well (50 μl/well) to obtain a final effector-to-target (E:T)ratio of 2:1 and a final volume of 200 μl per well. Then, 4 μl ofGloSensor (Promega #E1291) was added to each well (2% of final volume).Cells were incubated for 24 h at 37° C. in a humidified incubator. Atthe end of incubation time, luminescence was detected using TECAN Spark10M.

As shown in FIG. 18, the optimized CD3 binder P035-093 (P035)(=CD3_(opt)) had a similar activity on Jurkat NFAT cells uponcrosslinking as CD3_(orig).

Example 9

Generation of Bispecific Antibodies that Bind to Human HLA-G and toHuman CD3 (Anti-HLA-G/anti-CD3 Antibodies)

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany) The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany)

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. antibody heavy chainor antibody light chain) a transcription unit comprising the followingfunctional elements is used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain or MHC class I molecule), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to beexpressed the basic/standard mammalian expression plasmid contains

-   -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

Protein Determination

The protein concentration of purified polypeptides was determined bydetermining the optical density (OD) at 280 nm, using the molarextinction coefficient calculated on the basis of the amino acidsequence of the polypeptide.

Generation of Expression Plasmids for Recombinant Monoclonal BispecificAntibodies

The recombinant monoclonal antibody genes encode the respectiveimmunoglobulin heavy and light chains.

The expression plasmids for the transient expression monoclonal antibodymolecules comprised besides the immunoglobulin heavy or light chainexpression cassette an origin of replication from the vector pUC18,which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of a respective antibody heavy or light chaincomprised the following functional elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Transient Expression and Analytical Characterization

The recombinant production was performed by transient transfection ofHEK293 cells (human embryonic kidney cell line 293-derived) cultivatedin F17 Medium (Invitrogen Corp.). For the production of monoclonalantibodies, cells were co-transfected with plasmids containing therespective immunoglobulin heavy- and light chain. For transfection“293-Fectin” Transfection Reagent (Invitrogen) was used. Transfectionwas performed as specified in the manufacturer's instructions. Cellculture supernatants were harvested three to seven (3-7) days aftertransfection.

Supernatants were stored at reduced temperature (e.g. −80° C.).

General information regarding the recombinant expression of humanimmunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al.,Biotechnol. Bioeng. 75 (2001) 197-203.

Using the above described methods for recombinant DNA techniques, thegeneration of expression plasmids for recombinant monoclonal antibodiesand transient expression and analytical characterization, the followingbispecific antibodies that bind to human HLA-G and to human CD3 wereproduced and analyzed:

Bispecific Antibodies that Bind to Human HLA-G and to Human CD3(Anti-HLA-G/anti-CD3 Antibodies) (SEQ ID Nos of Variable Regions VH/VLand Hypervariable Regions (HVRs) of Antigen Binding Moieties/SitesBinding Human HLA-G and of Antigen Binding Moieties/Sites Binding HumanCD3):

Anti-HLA-G antigen binding site HVR-H1 HVR-H2 HVR-H3 HVR-L1 HVR-L2HVR-L3 VH VL HLA-G- SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID 0090-S32P NO: 1 NO: 2 NO: 3 NO: 23 NO: 5 NO: 6 NO: 7 NO: 24 Anti-CD3antigen binding site HVR-H1 HVR-H2 HVR-H3 HVR-L1 HVR-L2 HVR-L3 VH VLP035-093 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (P035)NO: 52 NO: 53 NO: 54 NO: 55 NO: 56 NO: 57 NO: 58 NO: 59 Clone 22 SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (Cl22) NO: 60 NO: 61NO: 62 NO: 63 NO: 64 NO: 65 NO: 66 NO: 67 V9 SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: 68 NO: 69 NO: 70 NO: 71 NO: 72 NO: 73NO: 74 NO: 75

“Clone 22 (abbreviated as “C122”)” is an optimized CD3 binder (see WO2020/127619); “P035-093 (abbreviated as “P035”) is another optimizedvariant CD3 binder; V9 is another CD3 binder described e.g. in Rodrigueset al., Int J Cancer Suppl (1992) 7, 45-50, and WO 1992/22653 (SEQ IDNOs 20 and 17 of WO 1992/22653 are the VH and VL sequences)

Bispecific Anti-HLA-G/Anti-CD3 T Cell Bispecific (TCB) Antibodies:

P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035-093 (P035)):

-   -   SEQ ID NO: 76 light chain 1 P1AF7977    -   SEQ ID NO: 77 light chain 2 P1AF7977    -   SEQ ID NO: 78 heavy chain 1 P1AF7977    -   SEQ ID NO: 79 heavy chain 2 P1AF7977

P1AF7978 (HLA-G-0090-VL-S32P/CD3 Clone 22 (C122)):

-   -   SEQ ID NO: 80 light chain 1 P1AF7978    -   SEQ ID NO: 81 light chain 2 P1AF7978    -   SEQ ID NO: 82 heavy chain 1 P1AF7978    -   SEQ ID NO: 83 heavy chain 2 P1AF7978

P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9):

-   -   SEQ ID NO: 84 light chain 1 P1AF7979    -   SEQ ID NO: 85 light chain 2 P1AF7979    -   SEQ ID NO: 86 heavy chain 1 P1AF7979    -   SEQ ID NO:87 heavy chain 2 P1AF7979

Example 10

Binding and Stability of Bispecific Anti-HLA-G/Anti-CD3 Antibody (T CellBispecific (TCB) Antibody) to HLA-G

Stability Under Stress

The three TCB molecules featuring the same HLA-G targeting binderHLAG-0090-VL-S32P and three different CD3e binders were stressed for 14days under two different conditions:

-   -   pH 6.0 20 mM His/HisCl, 140 mM NaCl; at 40° C. (His 40° C.)    -   pH 7.4 PBS; at 37° C. (PBS 37° C.).

Afterwards, the material was analysed using CD-SDS, SEC, and Surfaceplasmon resonance) SPR to investigate chemical degradation and possibleeffects on target binding. For reference, the stressed material wascompared with material kept under storage conditions:

-   -   pH 6.0 20 mM His/HisCl, 140 mM NaCl; frozen at −80° C. (Ref.)

The results are listed in the following tables:

P1AF7977 P1AF7978 P1AF7979 (HLA-G- (HLA-G- (HLA-G- 0090-VL- 0090-VL-0090-VL- S32P/CD3 S32P/CD3 S32P/ CD3 Parameter P035) Cl22) V9) CE-SDS[%] Ref. 95 95 96 (Caliper, non-reducing) His 40° C. 93 96 95 PBS 37° C.93 94 95 CE-SDS [%] Ref. 100 100 100 (Caliper, reducing) His 40° C. 100100 100 PBS 37° C. 100 100 100 SEC monomer Ref. 99 99 99 [%] His 40° C.97 98 98 PBS 37° C. 96 96 97 Thermal stability 64 64 68 (DLS T_(agg))P1AF7977 P1AF7978 P1AF7979 (HLA-G- (HLA-G- (HLA-G- 0090-VL- 0090-VL-0090-VL- S32P/CD3 S32P/CD3 S32P/CD3 P035) Cl22) V9) HLA-G CD3 HLA-G CD3HLA-G CD3 HLAG or CD3 Ref. 100 100 100 100 100 100 specific binding His40° C. 100  99  99  97  99  96 (respectivly) ± stress, PBS 37° C.  98 96  98  92  98  91 by Biacore RAC

All three TCBs are showing an acceptable stability profile with moderateloss of binding after stress. Furthermore, Thermal stability wasmeasured by DLS (T_(agg)) and it is in the normal range known for humanIgG. RAC Binding was determined by Surface plasmon resonance (Biacore)as described in Example 2.

Example 11

Binding of Bispecific Anti-HLA-G/Anti-CD3 Antibody (T Cell Bispecific(TCB) Antibody) to CD3 Expressed on T-Cells (as Assessed by FlowCytometry)

Briefly, 100 ml fresh blood was collected in Erlenmeyer flasks and mixedwith 100 ml of isolation buffer (PBS with 2% FBS and 2mM EDTA). 25 ml ofthe suspension was then transferred carefully over 15 ml of ficoll in a50 ml tube and centrifuged for 15 min at 800 g without brakes. The PBMClayer in the ficoll gradient was then transferred to a fresh 50 ml tubewith isolation buffer and centrifuged at 300 g for 10 min at 4° C. ThePBMCs were then washed twice and the cells were pooled in 10 ml ofisolation buffer. PBMCs were frozen at −80° C. until further use. Tcells were isolated from the PBMCs using EasySep negative selectionhuman T cell Isolation kit (Stem cell, #17951) as per manufacturer'sinstructions. Binding of HLA-G TCBs to T cells was then measured by flowcytometry. Briefly, 500 μl of T cells (5×105 cells) were added to eachFACS tube. T cells were washed in 2 ml of staining buffer (PBS with 2%FBS) and centrifuged at 300 g at 4° C. for 5 minutes. The HLA-G TCBswere diluted at different concentrations ranging from 5-0.05 μg/ml inmedium. T cells were then resuspended in 100 μl of HLA-G TCB dilutionand incubated for 30 min in the dark at 4° C. After washing once with 2ml staining buffer, cells were centrifuged at 300 g for 5 min and thenresuspended in 100 μl of secondary antibody dilution (Alexa Fluor 488labeled anti-human IgG, 1:200) for 30 min at 4° C. in the dark. T cellswere washed twice with 2 ml staining buffer and centrifuged at 300 g for5 min at 4° C. Finally cells were resuspended in 500 μl medium andbinding of HLA-G TCBs to T Cells was detected on BD LSR. The binding ofHLA-G TCBs P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978(HLA-G-0090-VL-S32P/CD3 C122) and P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9)to CD3 on T cells at different concentrations is illustrated in FIG. 7.

Example 12

Binding of Bispecific Anti-HLA-G/anti-CD3 Antibody (T Cell Bispecific(TCB) Antibody) to Natural or Recombinant HLA-G Expressed on Cells (asAssessed by Flow Cytometry)

Binding ability of anti HLA-G TCB mAb to HLA-G expressed on differentcells and cell lines was assessed by FACS analysis. Either the bindingto naturally HLA-G expressing JEG3 tumor cells or Skov3 transfectantsand respective parental, untransfected cells is described.

For flow cytometry analysis, cells were stained with anti HLA-G TCB mAbat 4° C. Briefly, 25 μl/well of each cell suspension (5×104 cells/well)was transferred into a polypropylene 96-Well V-bottom plate andprechilled in the fridge at 5° C. for 10 min Anti-HLA-G samples werediluted in staining buffer to a 2-fold starting concentration of 80μg/ml. A 4-fold serial dilution of the antibodies was performed and 25μl/well of the antibody solution was added to the prepared cells andincubated for 1 h at 5° C. Cells were washed twice with 200 μl/wellstaining buffer and centrifugation at 300 g for 5 min Cell pellets wereresuspended in 25 μl of staining buffer afterwards. For detectionfluorescent labeled anti-species antibody (donkey anti human IgG (H+L)conjugated to PE, Jackson Immuno Research #709-116-149) was diluted1:100 in staining buffer and 25 μl/well detection antibody was added tothe cell suspension. After a 1 hour incubation at 5° C. cells were againwashed twice with staining buffer, resuspended in 70 μl of stainingbuffer and measured at a FACS Canto II. Bispecific anti-HLA-G/anti-CD3antibodies (T cell bispecific (TCB) antibodies) P1AF7977(HLA-G-0090-VL-S32P/CD3 P035); P1AF7978 (HLA-G-0090-VL-S32P/CD3 C122)and P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9) showed binding to JEG3 cellsand SKOV3 cells, transfected with HLAG (see FIG. 8). The EC50 values forFACS binding are listed in the table below.

Cell binding EC50 (nM) JEG3 SKOV3 HLA-G P1AF7977 0.42 1.5 P1AF7978 0.120.15 P1AF7979 0.36 0.58

Example 13

ILT2 and -4 Binding Inhibition of Bispecific Anti-HLA-G/anti-CD3Antibody (T Cell Bispecific (TCB) Antibody)

The ELISA was set up by coating the Fc tagged ILT2 and ILT4 respectivelyto Maxisorp microtiter plates. After incubation and washing steps, therespective antibodies are added at a concentration of 100 nM. SolubleHis tagged monomeric, dimeric or trimeric HLA-G was added to the wells.After incubation and washing steps, detection of bound receptor wascarried out by anti-His-antibody-POD conjugates. Percentage inhibition(%) is calculated in comparison to values obtained from wells withILT2/4+HLA-G (mono-, di-, or Trimer) without anti HLA-G or ILT2/4antibodies (100% binding=0% inhibition) and shown in the followingtable.

% Binding inhibition (133 nM) ILT2 ILT4 P1AF7977 (HLA-G-0090-VL-S32P/CD3P035) 100 92 P1AF7978 (HLA-G-0090-VL-S32P/CD3 Cl22) 100 91 P1AF7979(HLA-G-0090-VL-S32P/CD3 V9) 100 95

Example 14

Bispecific Anti-HLA-G/anti-CD3 Antibody (T Cell Bispecific (TCB)Antibody) Mediated IFN Gamma Secretion by T Cells

Ability of anti HLA-G TCB to induce IFN gamma secretion by T cells inthe presence of HLA-G expressing tumor cells was tested using SKOV3cells transfected with recombinant HLA-G (SKOV3 HLA-G) and JEG3 cellsexpressing endogenous HLA-G. IFN gamma secretion was detected by Luminextechnology. For measurement of IFN gamma secretion by T cells after TCBtreatment, co-cultures of PBMCs and SKOV3HLA-G cells or JEG3 cells wereincubated with anti-HLA-G TCB. Briefly, PBMCs were isolated from humanperipheral blood by density gradient centrifugation using LymphocyteSeparating Medium Tubes (PAN #P04-60125). PBMCs and SKOV 3 HLA-G cellswere seeded at a ratio of 10:1 in 96-well U bottom plates. Theco-culture was then incubated with HLA-G-TCB at different concentrationsas shown in the figure (FIG. 9) and incubated for 24 h at 37° C. in anincubator with 5% Co2. On the next day, supernatants were collected andIFN gamma secretion was measured using Milliplex MAP kit (Luminextechnology) according to the manufacturer's instructions. Bispecificanti-HLA-G/anti-CD3 (T cell bispecific (TCB)) antibodies P1AF7977(HLA-G-0090-VL-S32P/CD3 P035); P1AF7978 (HLA-G-0090-VL-S32P/CD3 C122)and P1AF7979 (HLA-G-0090-VL-532P/CD3 V9) induced IFN gamma secretion byT cells (FIG. 9). The EC50 values are listed in the table below.

IFNgamma induction EC₅₀ (nM) JEG3 SKOV3 HLA-G P1AF7977 20 30 P1AF79782.2 2.6 P1AF7979 29 16

Example 15

Induction of T Cell Mediated Cytotoxicity/Tumor Cell Killing byBispecific Anti-HLA-G/Anti-CD3 Antibody (T Cell Bispecific (TCB)Antibody)

Ability of anti HLA-G TCB to induce T cell mediated cytotoxicity in thepresence of HLA-G expressing tumor cells was tested on SKOV3 cellstransfected with recombinant HLA-G (SKOV 3 HLA-G) and JEG3 cellsexpressing endogenous HLA-G. Cytotoxicity was detected by measuringCaspase 8 activation in cells after treatment with HLA-G TCB. Formeasurement of Caspase 8 activation after HLA-G/anti-CD3 antibody (TCB)treatment, co-cultures of PBMCs and SKOV3 HLA-G cells or JEG3 cells wereincubated with anti-HLA-G TCB for 24 hours and Caspase8 activation wasmeasured using the Caspase8Glo kit (Promega, #G8200). Briefly, PBMCswere isolated from human peripheral blood by density gradientcentrifugation using Lymphocyte Separating Medium Tubes (PAN#P04-60125). PBMC's and SKOV3 HLA-G or JEG3 cells were seeded at a ratioof 10:1 (100 μl per well) in black clear bottom 96-well plates. Theco-culture was then incubated with HLA-G-TCB at different concentrationsas shown in the figure (FIG. 10) and incubated for 24 h or 48 h at 37°C. in an incubator with 5% Co2. On the next day, 100 μl of Caspase8 Glosubstrate was added to each well and placed on a shaker for 1 hour atroom temperature. The luminescence was measured on a BioTek Synergy 2machine. The relative luminescence units (RLUs) correspond to theCaspase8 activation/cytotoxicity are plotted in the graph (FIG. 10).Bispecific anti-HLA-G/anti-CD3 (T cell bispecific (TCB)) antibodiesP1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978 (HLA-G-0090-VL-S32P/CD3C122) and P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9) induced T cell mediatedcytotoxicity/tumor cell killing. The EC50 values of the tumor cellkilling are listed in the table below.

T cell mediated cytotoxicity induction EC₅₀ (nM) JEG3 SKOV3 HLA-GP1AF7977 12 1.4 P1AF7978 2.6 1.36 P1AF7979 4.6 1

Example 16

In Vivo Anti-Tumor Efficacy of Bispecific Anti-HLA-G/Anti-CD3 (T CellBispecific (TCB)) Antibody in Humanized NSG Mice Bearing SKOV3 HumanOvarian Carcinoma Transfected with Recombinant HLA-G (SKOV3 HLA-G)

Humanized NSG (NOD/scid/IL-2Rγnull humanized with CD34+ cord blood cellsby Jackson Laboratories, US) mice (n=15) were injected subcutaneouslywith 5×10⁶ SKOV3 HLA-G cells in a total volume of 100 μl. Once thetumors reached an average volume of 200 mm3, mice were randomized andtreated weekly with bispecific anti-HLA-G/anti-CD3 (T cell bispecific(TCB)) antibody (P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035)) (5 mg/kg)weekly. As a control, one group of mice received weekly i.v. injectionsof histidine buffer (vehicle). Tumor volume was measured twice weeklyuntil study termination. The results of the experiment are shown in FIG.11. Results show tumor volume data (Median and Inter quartile range(IQR)) measured by caliper in the two study groups. Theanti-HLA-G/anti-CD3 T cell bispecific (TCB) antibody P1AF7977 showedstrong tumor growth inhibition/tumor regression in the SKOV3-HLA-G tumormodel.

Example 17

Dose-Response Study Bispecific Anti-HLA-G/Anti-CD3 (T Cell Bispecific(TCB)) Antibody in Humanized NSG Mice Bearing Human Breast Cancer PDXTumors (BC004)

Humanized NSG (NOD/scid/IL-2Rγnull humanized by intravenous injection of1×10⁵ CD34+ cord blood cells per mouse) mice were injected with 2×10⁶BC004 breast cancer cells in total volume of 50 μL PBS into theintra-mammary fat pad. Once the tumors reached an average volume ofapproximately 200 mm3, mice were randomized (n=15 animals per group) andtreated weekly with bispecific anti-HLA-G/anti-CD3 (T cell bispecific(TCB)) antibody (P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035)) with threedifferent doses (5 mg/kg, 2.5 mg/kg, 0.5mg/kg). As a control, one groupof mice received weekly i.v. injections of histidine buffer (vehicle).Tumor volume was determined twice weekly via caliper measurement. Theresults of the experiment shown in FIG. 12 demonstrates tumor volumedata (Median and Inter quartile range (IQR)). All three doses ofanti-HLA-G/anti-CD3 T cell bispecific (TCB) antibody showed strong tumorgrowth inhibition/tumor regression in the BC004 tumor model. The highestdose (5 mg/kg) shows slightly higher efficacy compared to the 2.5 mg/kgand 0.5 mg/kg treatment groups.

Example 18

Induction of T Cell Activation in the Presence of HLA-G Expressing TumorCells was Tested on SKOV3 Cells Transfected with Recombinant HLA-G(SKOV3 HLA-G) by Bispecific Anti-HLA-G/Anti-CD3 Antibody (T CellBispecific (TCB) Antibody)

Ability of anti HLA-G/anti CD3 TCB to activate T cells in the presenceof HLA-G expressing tumor cells was tested on SKOV3 cells transfectedwith recombinant HLA-G (SKOV3 HLA-G). Activation of T cells was assessedby FACS analysis of cell surface activation markers CD25 and earlyactivation marker CD69 on T cells. Briefly, Peripheral Blood MononuclearCells (PBMCs) are isolated from human peripheral blood by densitygradient centrifugation using Lymphocyte Separating Medium Tubes (PAN#P04-60125). PBMC's and SKOV3 HLA-G cells are seeded at a ratio of 10:1in 96-well U bottom plates. The co-culture was then incubated withanti-HLA-G/anti-CD3 (T cell bispecific (TCB)) antibody (P1AF7977(HLA-G-0090-VL-S32P/CD3 P035)) (0.01 nM) and incubated for 24 h at 37°C. in an incubator with 5% Co2. On the next day, expression of CD25 andCD69 was measured by flow cytometry.

For flow cytometry analysis, cells are stained with with PerCP-Cy5.5Mouse Anti-Human CD8 (BD Pharmingen #565310), PE-Cy7 Mouse Anti-HumanCD4 (Biologend #317414), FITC Mouse Anti-Human CD25 (Biolegend #356106)and APC Mouse Anti-Human CD69 (BD Pharmingen #555533) at 4° C. Briefly,antibodies are diluted to a 2-fold concentration and 25 μl of antibodydilution are added in each well with 25 μl of pre-washed co-cultures.Cells are stained for 30 min at 4° C. and washed twice with 200 μl/wellstaining buffer and centrifugation at 300 g for 5 min Cell pellets areresuspended in 200 μl of staining buffer and stained with DAPI for livedead discrimination at a final concentration of 2 μg/ml. Samples arethen measured using BD LSR flow cytometer. Data analysis was performedusing FlowJo V.10.1 software. FIG. 14 shows the induction of T cellactivation by bispecific anti-HLA-G/anti-CD3 antibody P1AF7977(HLA-G-0090-VL-S32P/CD3 P035 in the presence of SKOV3 HLAG cells.

1. An antibody that binds to human HLA-G comprising A) (a) a VH domaincomprising (i) CDR-H1 comprising an amino acid sequence of SEQ ID NO:1,(ii) CDR-H2 comprising an amino acid sequence of SEQ ID NO:2, and (iii)CDR-H3 comprising an amino acid sequence of SEQ ID NO:3; and (b) a VLdomain comprising (i) CDR-L1 comprising an amino acid sequence of SEQ IDNO:23; (ii) CDR-L2 comprising an amino acid sequence of SEQ ID NO:5 and(iii) CDR-L3 comprising an amino acid sequence of SEQ ID NO:6, or B) (a)a VH domain comprising (i) CDR-H1 comprising an amino acid sequence ofSEQ ID NO:1, (ii) CDR-H2 comprising an amino acid sequence of SEQ IDNO:2, and (iii) CDR-H3 comprising an amino acid sequence of SEQ ID NO:3;and (b) a VL domain comprising (i) CDR-L1 comprising an amino acidsequence of SEQ ID NO:25; (ii) CDR-L2 comprising an amino acid sequenceof SEQ ID NO:5 and (iii) CDR-L3 comprising an amino acid sequence of SEQID NO:6.
 2. The antibody according to claim 1, wherein the antibody A)comprises a VH domain comprising an amino acid sequence of SEQ ID NO:7and a VL domain comprising an amino acid sequence of SEQ ID NO:24; or B)comprises a VH domain comprising an amino acid sequence of SEQ ID NO:7and a VL domain comprising an amino acid sequence of SEQ ID NO:26. 3.The antibody of claim 1, wherein the antibody comprises a Fc domain ofhuman origin. 4.-25. (canceled)
 26. The antibody of claim 3, wherein theFc domain of human origin is an IgG isotype.
 27. The antibody of claim26, wherein the IgG is an IgG1 isotype.
 28. The antibody of claim 27,wherein the antibody comprises a constant region of human origin,comprising a human CH1, CH2, CH3 and/or CL domain.
 29. The antibody ofclaim 1, wherein the antibody does not cross-react with: a) a modifiedhuman HLA-G β2M MHC I complex, wherein the HLA-G specific amino acidshave been replaced by HLA-A consensus amino acids, the complexcomprising SEQ ID NO:40; b) a mouse H2Kd β2M MHC I complex comprisingSEQ ID NO:41; or c) a rat RT1A β2M MHC I complex comprising SEQ IDNO:43.
 30. The antibody of claim 1, wherein the antibody: a) inhibitsILT2 binding to HLA-G expressed on JEG3 cells (ATCC No. HTB36); or b)binds to HLA-G expressed on JEG3 cells (ATCC No. HTB36) and inhibitsILT2 binding to HLA-G expressed on JEG-3 cells (ATCC No. HTB36).
 31. Theantibody of claim 1, wherein the antibody is a multi-specific antibody.32. One or more isolated nucleic acids encoding the antibody of claim 1.33. A host cell comprising the one or more nucleic acids of claim 32.34. The host cell of claim 33, wherein the host cell is an eukaryoticcell.
 35. A method of producing the antibody encoded by the one or morenucleic acids of the host cell of claim 33, comprising culturing thehost cell so that the antibody is produced.
 36. The method of claim 35,further comprising recovering the antibody from the host cell.
 37. Apharmaceutical formulation comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 38. A method of treating anindividual having cancer comprising administering to the individual aneffective amount of the antibody of claim
 1. 39. A bispecific antibody,comprising a first antigen binding moiety that binds to human HLA-G andcomprises: A) (a) a VH domain comprising (i) CDR-H1 comprising an aminoacid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising an amino acidsequence of SEQ ID NO:2, and (iii) CDR-H3 comprising an amino acidsequence of SEQ ID NO:3; and (b) a VL domain comprising (i) CDR-L1comprising an amino acid sequence of SEQ ID NO:23; (ii) CDR-L2comprising an amino acid sequence of SEQ ID NO:5 and (iii) CDR-L3comprising an amino acid sequence of SEQ ID NO:6, or B) (a) a VH domaincomprising (i) CDR-H1 comprising an amino acid sequence of SEQ ID NO:1,(ii) CDR-H2 comprising an amino acid sequence of SEQ ID NO:2, and (iii)CDR-H3 comprising an amino acid sequence of SEQ ID NO:3; and (b) a VLdomain comprising (i) CDR-L1 comprising an amino acid sequence of SEQ IDNO:25; (ii) CDR-L2 comprising an amino acid sequence of SEQ ID NO:5 and(iii) CDR-L3 comprising an amino acid sequence of SEQ ID NO:6; and asecond antigen binding moiety that binds to human CD3 and comprises: C)(a) a VH domain comprising (i) CDR-H1 comprising an amino acid sequenceof SEQ ID NO:52, (ii) CDR-H2 comprising an amino acid sequence of SEQ IDNO:53, and (iii) CDR-H3 comprising an amino acid sequence of SEQ IDNO:54; and (b) a VL domain comprising (i) CDR-L1 comprising an aminoacid sequence of SEQ ID NO:55; (ii) CDR-L2 comprising an amino acidsequence of SEQ ID NO:56 and (iii) CDR-L3 comprising an amino acidsequence of SEQ ID NO:57, or D) (a) a VH domain comprising (i) CDR-H1comprising an amino acid sequence of SEQ ID NO:60, (ii) CDR-H2comprising an amino acid sequence of SEQ ID NO:61, and (iii) CDR-H3comprising an amino acid sequence of SEQ ID NO:62; and (b) a VL domaincomprising (i) CDR-L1 comprising an amino acid sequence of SEQ ID NO:63;(ii) CDR-L2 comprising an amino acid sequence of SEQ ID NO:64 and (iii)CDR-L3 comprising an amino acid sequence of SEQ ID NO:65, or E) (a) a VHdomain comprising (i) CDR-H1 comprising an amino acid sequence of SEQ IDNO:68, (ii) CDR-H2 comprising an amino acid sequence of SEQ ID NO:69,and (iii) CDR-H3 comprising an amino acid sequence of SEQ ID NO:70; and(b) a VL domain comprising (i) CDR-L1 comprising an amino acid sequenceof SEQ ID NO:71; (ii) CDR-L2 comprising an amino acid sequence of SEQ IDNO:72 and (iii) CDR-L3 comprising an amino acid sequence of SEQ IDNO:73.
 40. The bispecific antibody according to claim 39, wherein thefirst antigen binding moiety comprises: A) a VH domain comprising anamino acid sequence of SEQ ID NO:7 and a VL domain comprising an aminoacid sequence of SEQ ID NO:24; or B) a VH domain comprising an aminoacid sequence of SEQ ID NO:7 and a VL domain comprising an amino acidsequence of SEQ ID NO:26, and wherein the second antigen binding moietycomprises: C) a VH domain comprising an amino acid sequence of SEQ IDNO:58 and a VL domain comprising an amino acid sequence of SEQ ID NO:59;or D) a VH domain comprising an amino acid sequence of SEQ ID NO:66 anda VL domain comprising an amino acid sequence of SEQ ID NO:67; or E) aVH domain comprising an amino acid sequence of SEQ ID NO:74 and a VLdomain comprising an amino acid sequence of SEQ ID NO:75.
 41. Thebispecific antibody according to claim 40, wherein the first antigenbinding moiety comprises a VH domain comprising an amino acid sequenceof SEQ ID NO:7 and a VL domain comprising an amino acid sequence of SEQID NO:24; and wherein the second antigen binding moiety comprises a VHdomain comprising an amino acid sequence of SEQ ID NO:58 and a VL domaincomprising an amino acid sequence of SEQ ID NO:59.
 42. The bispecificantibody according to claim 40, wherein the first antigen binding moietycomprises a VH domain comprising an amino acid sequence of SEQ ID NO:7and a VL domain comprising an amino acid sequence of SEQ ID NO:24; andwherein the second antigen binding moiety comprises a VH domaincomprising an amino acid sequence of SEQ ID NO:66 and a VL domaincomprising an amino acid sequence of SEQ ID NO:67.
 43. The bispecificantibody according to claim 40, wherein the first antigen binding moietycomprises a VH domain comprising an amino acid sequence of SEQ ID NO:7and a VL domain comprising an amino acid sequence of SEQ ID NO:24; andwherein the second antigen binding moiety comprises a VH domaincomprising an amino acid sequence of SEQ ID NO:74 and a VL domaincomprising an amino acid sequence of SEQ ID NO:75.
 44. The bispecificantibody of claim 39, wherein the bispecific antibody shows: a)inhibition of ILT2 or ILT4 binding to HLA-G; b) antibody-mediated IFNgamma secretion by T cells on i) SKOV3 cells transfected withrecombinant HLA-G (SKOV3 HLA-G); or ii) JEG3 cells expressing endogenousHLA-G; c) T cell-mediated cytotoxicity or tumor cell killing on i) SKOV3cells transfected with recombinant HLA-G (SKOV 3HLA-G); or ii) JEG3cells expressing endogenous HLA-G; d) in vivo anti-tumor efficacy ortumor regression in humanized NSG mice bearing SKOV3 human ovariancarcinoma transfected with recombinant HLA-G (SKOV3 HLA-G); or e) invivo anti-tumor efficacy or tumor in humanized NSG mice bearing humanbreast cancer PDX tumors (BC004).
 45. One or more isolated nucleic acidsencoding the bispecific antibody of claim
 39. 46. A host cell comprisingthe one or more nucleic acids of claim
 45. 47. The host cell of claim46, wherein the host cell is an eukaryotic cell.
 48. A method ofproducing the bispecific antibody encoded by the one or more nucleicacids of the host cell of claim 46, comprising culturing the host cellso that the bispecific antibody is produced.
 49. The method of claim 48,further comprising recovering the bispecific antibody from the hostcell.
 50. A pharmaceutical formulation comprising the bispecificantibody of claim 39 and a pharmaceutically acceptable carrier.
 51. Amethod of treating an individual having cancer comprising administeringto the individual an effective amount of the bispecific antibody ofclaim 39.